Differential knockout of an allele of a heterozygous bestrophin 1 gene

ABSTRACT

RNA molecules comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and compositions, methods, and uses thereof.

This application claims the benefit of U.S. Provisional Application No. 62/680,482, filed Jun. 4, 2018 and U.S. Provisional Application No. 62/591,365, filed Nov. 28, 2017, the contents of each of which are hereby incorporated by reference.

Throughout this application, various publications are referenced, including referenced in parenthesis. The disclosures of all publications mentioned in this application in their entireties are hereby incorporated by reference into this application in order to provide additional description of the art to which this invention pertains and of the features in the art which can be employed with this invention.

REFERENCE TO SEQUENCE LISTING

This application incorporates-by-reference nucleotide sequences which are present in the filed named “220803_90240-A_Substitute_Sequence_Listing_AWG.txt”, which is 552 kilobytes in size, and which was created on May 23, 2022 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, which is contained in the text file filed Aug. 3, 2022 as part of this application.

BACKGROUND OF INVENTION

There are several classes of DNA variation in the human genome, including insertions and deletions, differences in the copy number of repeated sequences, and single nucleotide polymorphisms (SNPs). A SNP is a DNA sequence variation occurring when a single nucleotide (adenine (A), thymine (T), cytosine (C), or guanine (G)) in the genome differs between human subjects or paired chromosomes in an individual. Over the years, the different types of DNA variations have been the focus of the research community either as markers in studies to pinpoint traits or disease causation or as potential causes of genetic disorders.

A genetic disorder is caused by one or more abnormalities in the genome. Genetic disorders may be regarded as either “dominant” or “recessive.” Recessive genetic disorders are those which require two copies (i.e., two alleles) of the abnormal/defective gene to be present. In contrast, a dominant genetic disorder involves a gene or genes which exhibit(s) dominance over a normal (functional/healthy) gene or genes. As such, in dominant genetic disorders only a single copy (i.e., allele) of an abnormal gene is required to cause or contribute to the symptoms of a particular genetic disorder. Such mutations include, for example, gain-of-function mutations in which the altered gene product possesses a new molecular function or a new pattern of gene expression. Other examples include dominant negative mutations, which have a gene product that acts antagonistically to the wild-type allele.

Best Vitelliform Macular Dystrophy

Best vitelliform macular dystrophy is commonly a slowly progressive macular dystrophy with onset generally in childhood and sometimes in later teenage years. Affected individuals may initially have normal vision followed by decreased central visual acuity and metamorphopsia. Individuals retain normal peripheral vision and dark adaptation. Best vitelliform macular dystrophy is commonly inherited in an autosomal dominant manner, although, autosomal recessive inheritance has also been reported. Mutations in bestrophin 1 gene (BEST1) have been associated with autosomal dominant Best vitelliform macular dystrophy.

SUMMARY OF THE INVENTION

Disclosed is an approach for knocking out the expression of a dominant-mutated allele by disrupting the dominant-mutated allele or degrading the resulting mRNA.

The present disclosure provides a method for utilizing at least one naturally occurring nucleotide difference or polymorphism (e.g., single nucleotide polymorphism (SNP)) for distinguishing/discriminating between two alleles of a gene, one allele bearing a mutation such that it encodes a mutated protein causing a disease phenotype (“mutated allele”), and the other allele encoding for a functional protein (“functional allele”). In some embodiments, the method further comprises the step of knocking out expression of the mutated protein and allowing expression of the functional protein.

According to embodiments of the present invention, there is provided a first RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010.

According to embodiments of the present invention, there is provided a first RNA molecule comprising a guide sequence portion having 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010.

According to some embodiments of the present invention, there is provided a composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to some embodiments of the present invention, there is provided a method for inactivating a mutant BEST1 allele in a cell, the method comprising delivering to the cell a composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to some embodiments of the present invention, there is provided a method for treating Best Vitelliform Macular Dystrophy, the method comprising delivering to a subject having Best Vitelliform Macular Dystrophy, a composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to some embodiments of the present invention, there is provided use of a composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease for inactivating a mutant BEST1 allele in a cell, comprising delivering to the cell the composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to embodiments of the present invention, there is provided a medicament comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease for use in inactivating a mutant BEST1 allele in a cell, wherein the medicament is administered by delivering to the cell the composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to some embodiments of the present invention, there is provided use of a composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease for treating ameliorating or preventing Best Vitelliform Macular Dystrophy, comprising delivering to a subject having or at risk of having Best Vitelliform Macular Dystrophy the composition of comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to some embodiments of the present invention, there is provided a medicament comprising the composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease for use in treating ameliorating or preventing Best Vitelliform Macular Dystrophy, wherein the medicament is administered by delivering to a subject having or at risk of having Best Vitelliform Macular Dystrophy the composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to some embodiments of the present invention, there is provided a kit for inactivating a mutant BEST1 allele in a cell, comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010, a CRISPR nuclease, and/or a tracrRNA molecule; and instructions for delivering the RNA molecule; CRISPR nuclease, and/or the tracrRNA to the cell.

According to some embodiments of the present invention, there is provided a kit for treating Best Vitelliform Macular Dystrophy in a subject, comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010, a CRISPR nuclease, and/or a tracrRNA molecule; and instructions for delivering the RNA molecule; CRISPR nuclease, and/or the tracrRNA to a subject having or at risk of having Best Vitelliform Macular Dystrophy.

DETAILED DESCRIPTION Definitions

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a,” “an” and “at least one” are used interchangeably in this application.

For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.

In the description and claims of the present application, each of the verbs, “comprise,” “include” and “have” and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb. Other terms as used herein are meant to be defined by their well-known meanings in the art.

The “guide sequence portion” of an RNA molecule refers to a nucleotide sequence that is capable of hybridizing to a specific target DNA sequence, e.g., the guide sequence portion has a nucleotide sequence which is fully complementary to said target DNA sequence. In some embodiments, the guide sequence portion is 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length, or approximately 17-24, 18-22, 19-22, 18-20, or 17-20 nucleotides in length. The guide sequence portion may be part of an RNA molecule that can form a complex with a CRISPR nuclease with the guide sequence portion serving as the DNA targeting portion of the CRISPR complex. When the DNA molecule having the guide sequence portion is present contemporaneously with the CRISPR molecule the RNA molecule is capable of targeting the CRISPR nuclease to the specific target DNA sequence. Each possibility represents a separate embodiment. An RNA molecule can be custom designed to target any desired sequence.

In embodiments of the present invention, an RNA molecule comprises a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010, 1-714, or 715-3010.

As used herein, “contiguous nucleotides” set forth in a SEQ ID NO refers to nucleotides in a sequence of nucleotides in the order set forth in the SEQ ID NO without any intervening nucleotides.

In embodiments of the present invention, the guide sequence portion may be 20 nucleotides in length and consists of 20 nucleotides in the sequence of 20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010. In embodiments of the present invention, the guide sequence portion may be less than 20 nucleotides in length. For example, in embodiments of the present invention the guide sequence portion may be 17, 18, or 19 nucleotides in length. In such embodiments the guide sequence portion may consist of 17, 18, or 19 nucleotides, respectively, in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010. For example, a guide sequence portion having 17 nucleotides in the sequence of 17 contiguous nucleotides set forth in SEQ ID NO: 1 may consist of any one of the following nucleotide sequences (nucleotides excluded from the contiguous sequence are marked in strike-through):

-   -   AUCCGUCAGGUUAAACUCCA (SEQ ID NO: 1)     -   17 nucleotide guide sequence 1: CGUCAGGUUAAACUCCA (SEQ ID NO:         3011)     -   17 nucleotide guide sequence 2: CCGUCAGGUUAAACUCC (SEQ ID NO:         3012)     -   17 nucleotide guide sequence 3: UCCGUCAGGUUAAACUC (SEQ ID NO:         3013)     -   17 nucleotide guide sequence 4: AUCCGUCAGGUUAAACU (SEQ ID NO:         3014)

In embodiments of the present invention, the guide sequence portion may be greater than 20 nucleotides in length. For example, in embodiments of the present invention the guide sequence portion may be 21, 22, 23, or 24 nucleotides in length. In such embodiments the guide sequence portion comprises 20 nucleotides in the sequence of 20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and additional nucleotides fully complimentary to a nucleotide or sequence of nucleotides adjacent to the 3′ end of the target sequence, 5′ end of the target sequence, or both.

In embodiments of the present invention a CRISPR nuclease and an RNA molecule comprising a guide sequence portion form a CRISPR complex that binds to a target DNA sequence to effect cleavage of the target DNA sequence. CRISPR nucleases, e.g. Cpf1, may form a CRISPR complex comprising a CRISPR nuclease and RNA molecule without a further tracrRNA molecule. Alternatively, CRISPR nucleases, e.g. Cas9, may form a CRISPR complex between the CRISPR nuclease, an RNA molecule, and a tracrRNA molecule.

In embodiments of the present invention, the RNA molecule may further comprise the sequence of a tracrRNA molecule. Such embodiments may be designed as a synthetic fusion of the guide portion of the RNA molecule and the trans-activating crRNA (tracrRNA). (See Jinek (2012) Science). Embodiments of the present invention may also form CRISPR complexes utilizing a separate tracrRNA molecule and a separate RNA molecule comprising a guide sequence portion. In such embodiments the tracrRNA molecule may hybridize with the RNA molecule via basepairing and may be advantageous in certain applications of the invention described herein.

The term “tracr mate sequence” refers to a sequence sufficiently complementary to a tracrRNA molecule so as to hybridize to the tracrRNA via basepairing and promote the formation of a CRISPR complex. (See e.g., U.S. Pat. No. 8,906,616). In embodiments of the present invention, the RNA molecule may further comprise a portion having a tracr mate sequence.

A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.

“Eukaryotic” cells include, but are not limited to, fungal cells (such as yeast), plant cells, animal cells, mammalian cells and human cells.

The term “nuclease” as used herein refers to an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acid. A nuclease may be isolated or derived from a natural source. The natural source may be any living organism. Alternatively, a nuclease may be a modified or a synthetic protein which retains the phosphodiester bond cleaving activity. Gene modification can be achieved using a nuclease, for example a CRISPR nuclease.

Embodiments

The present disclosure provides a method for utilizing at least one naturally occurring nucleotide difference or polymorphism (e.g., single nucleotide polymorphism (SNP)) for distinguishing/discriminating between two alleles of a gene, one allele bearing a mutation such that it encodes a mutated protein causing a disease phenotype (“mutated allele”), and the other allele encoding for a functional protein (“functional allele”). The method further comprises the step of knocking out expression of the mutated protein and allowing expression of the functional protein. In some embodiments, the method is for treating, ameliorating, or preventing a dominant negative genetic disorder.

According to embodiments of the present invention, there is provided a first RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010.

According to embodiments of the present invention, there is provided a first RNA molecule comprising a guide sequence portion having 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010.

According embodiments of the present invention, an RNA molecule may further comprise a portion having a sequence which binds to a CRISPR nuclease.

According to embodiments of the present invention, the sequence which binds to a CRISPR nuclease is a tracrRNA sequence.

According to embodiments of the present invention, an RNA molecule may further comprise a portion having a tracr mate sequence.

According to embodiments of the present invention, an RNA molecule may further comprise one or more linker portions.

According to embodiments of the present invention, an RNA molecule may be up to 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, or 100 nucleotides in length. Each possibility represents a separate embodiment. In embodiments of the present invention, the RNA molecule may be 17 up to 300 nucleotides in length, 100 up to 300 nucleotides in length, 150 up to 300 nucleotides in length, 200 up to 300 nucleotides in length, 100 to 200 nucleotides in length, or 150 up to 250 nucleotides in length. Each possibility represents a separate embodiment.

According to some embodiments of the present invention, there is provided a composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to embodiments of the present invention, the composition may comprise a second RNA molecule comprising a guide sequence portion.

According to embodiments of the present invention, the guide sequence portion of the second RNA molecule comprises 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010.

According to embodiments of the present invention, the 17-20 nucleotides of the guide sequence portion of the second RNA molecule are in a different sequence from the sequence of the guide sequence portion of the first RNA molecule

Embodiments of the present invention may comprise a tracrRNA molecule.

According to some embodiments of the present invention, there is provided a method for inactivating a mutant BEST1 allele in a cell, the method comprising delivering to the cell a composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to some embodiments of the present invention, there is provided a method for treating Best Vitelliform Macular Dystrophy, the method comprising delivering to a subject having Best Vitelliform Macular Dystrophy a composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to embodiments of the present invention, the composition comprises a second RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010.

According to embodiments of the present invention, the 17-20 nucleotides of the guide sequence portion of the second RNA molecule are in a different sequence from the sequence of the guide sequence portion of the first RNA molecule

According to embodiments of the present invention, the CRISPR nuclease and the RNA molecule or RNA molecules are delivered to the subject and/or cells substantially at the same time or at different times.

According to embodiments of the present invention, the tracrRNA is delivered to the subject and/or cells substantially at the same time or at different times as the CRISPR nuclease and RNA molecule or RNA molecules.

According to embodiments of the present invention, the first RNA molecule targets a SNP or disease-causing mutation in an exon or promoter of a mutated allele, and wherein the second RNA molecule targets a SNP in the same or a different exon of the mutated allele, a SNP in an intron, or a sequence in an intron present in both the mutated or functional allele.

According to embodiments of the present invention, the first RNA molecule or the first and the second RNA molecules target a SNP in the promoter region, the start codon, or the untranslated region (UTR) of a mutated allele.

According to embodiments of the present invention, the first RNA molecule or the first and the second RNA molecules targets at least a portion of the promoter and/or the start codon and/or a portion of the UTR of a mutated allele.

According to embodiments of the present invention, the first RNA molecule targets a portion of the promoter, a first SNP in the promoter, or a SNP upstream to the promoter of a mutated allele and the second RNA molecule is targets a second SNP, which is downstream of the first SNP, and is in the promoter, in the UTR, or in an intron or in an exon of a mutated allele.

According to embodiments of the present invention, the first RNA molecule targets a SNP in the promoter, upstream of the promoter, or the UTR of a mutated allele and the second RNA molecule is designed to target a sequence which is present in an intron of both the mutated allele and the functional allele.

According to embodiments of the present invention, the first RNA molecule targets a sequence upstream of the promotor which is present in both a mutated and functional allele and the second RNA molecule targets a SNP or disease-causing mutation in any location of the gene.

According to embodiments of the present invention, there is provided a method comprising removing an exon containing a disease-causing mutation from a mutated allele, wherein the first RNA molecule or the first and the second RNA molecules target regions flanking an entire exon or a portion of the exon.

According to embodiments of the present invention, there is provided a method comprising removing multiple exons, the entire open reading frame of a gene, or removing the entire gene.

According to embodiments of the present invention, the first RNA molecule targets a SNP or disease-causing mutation in an exon or promoter of a mutated allele, and wherein the second RNA molecule targets a SNP in the same or a different exon of the mutated allele, a SNP in an intron, or a sequence in an intron present in both the mutated or functional allele.

According to embodiments of the present invention, the first RNA molecule or the first and the second RNA molecules target an alternative splicing signal sequence between an exon and an intron of a mutant allele.

According to embodiments of the present invention, the second RNA molecule targets a sequence present in both a mutated allele and a functional allele.

According to embodiments of the present invention, the second RNA molecule targets an intron.

According to embodiments of the present invention, there is provided a method comprising subjecting the mutant allele to insertion or deletion by an error prone non-homologous end joining (NHEJ) mechanism, generating a frameshift in the mutated allele's sequence.

According to embodiments of the present invention, the frameshift results in inactivation or knockout of the mutated allele.

According to embodiments of the present invention, the frameshift creates an early stop codon in the mutated allele.

According to embodiments of the present invention, the frameshift results in nonsense-mediated mRNA decay of the transcript of the mutant allele.

According to embodiments of the present invention, the inactivating or treating results in a truncated protein encoded by the mutated allele and a functional protein encoded by the functional allele.

According to some embodiments of the present invention, there is provided use of a composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease inactivating a mutant BEST1 allele in a cell, comprising delivering to the cell the RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and the CRISPR nuclease.

According to embodiments of the present invention, there is provided a medicament comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease for use in inactivating a mutant BEST1 allele in a cell, wherein the medicament is administered by delivering to the cell the composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to some embodiments of the present invention, there is provided use of a composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease for treating ameliorating or preventing Best Vitelliform Macular Dystrophy, comprising delivering to a subject having or at risk of having Best Vitelliform Macular Dystrophy the composition of comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to some embodiments of the present invention, there is provided a medicament comprising the composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease for use in treating ameliorating or preventing Best Vitelliform Macular Dystrophy, wherein the medicament is administered by delivering to a subject having or at risk of having Best Vitelliform Macular Dystrophy: the composition comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010 and a CRISPR nuclease.

According to some embodiments of the present invention, there is provided a kit for inactivating a mutant BEST1 allele in a cell, comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010, a CRISPR nuclease, and/or a tracrRNA molecule; and instructions for delivering the RNA molecule; CRISPR nuclease, and/or the tracrRNA to the cell.

According to some embodiments of the present invention, there is provided a kit for treating Best Vitelliform Macular Dystrophy in a subject, comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010, a CRISPR nuclease, and/or a tracrRNA molecule; and instructions for delivering the RNA molecule; CRISPR nuclease, and/or the tracrRNA to a subject having or at risk of having Best Vitelliform Macular Dystrophy.

In embodiments of the present invention, the RNA molecule comprises a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-714, SEQ ID NOs: 715-3010, or SEQ ID NOs 1-3010.

The compositions and methods of the present disclosure may be utilized for treating, preventing, ameliorating, or slowing progression of Best Vitelliform Macular Dystrophy.

In some embodiments, a mutated allele is deactivated by delivering to a cell an RNA molecule which targets a SNP in the promoter region, the start codon, or the untranslated region (UTR) of the mutated allele.

In some embodiments, a mutated allele is inactivated by removing at least a portion of the promoter and/or removing the start codon and/or a portion of the UTR. In some embodiments, the method of deactivating a mutated allele comprises removing at least a portion of the promoter. In such embodiments one RNA molecule may be designed for targeting a first SNP in the promoter or upstream to the promoter and another RNA molecule is designed to target a second SNP, which is downstream of the first SNP, and is in the promoter, in the UTR, or in an intron or in an exon. Alternatively, one RNA molecule may be designed for targeting a SNP in the promoter, or upstream of the promoter, or the UTR and another RNA molecule is designed to target a sequence which is present in an intron of both the mutated allele and the functional allele. Alternatively, one RNA molecule may be designed for targeting a sequence upstream of the promotor which is present in both the mutated and functional allele and the other guide is designed to target a SNP or disease-causing mutation in any location of the gene e.g., in an exon, intron, UTR, or downstream of the promoter.

In some embodiments, the method of deactivating a mutated allele comprises an exon skipping step comprising removing an exon containing a disease-causing mutation from the mutated allele. Removing an exon containing a disease-causing mutation in the mutated allele requires two RNA molecules which target regions flanking the entire exon or a portion of the exon. Removal of an exon containing the disease-causing mutation may be designed to eliminate the disease-causing action of the protein while allowing for expression of the remaining protein product which retains some or all of the wild-type activity. As an alternative to single exon skipping, multiple exons, the entire open reading frame or the entire gene can be excised using two RNA molecules flanking the region desired to be excised.

In some embodiments, the method of deactivating a mutated allele comprises delivering two RNA molecules to a cell, wherein one RNA molecule targets a SNP or disease-causing mutation in an exon or promoter of the mutated allele, and wherein the other RNA molecule targets a SNP in the same or a different exon of the mutated allele, a SNP in an intron, or a sequence in an intron present in both the mutated or functional allele.

In some embodiments, an RNA molecule is used to target a CRISPR nuclease to an alternative splicing signal sequence between an exon and an intron of a mutant allele, thereby destroying the alternative splicing signal sequence in the mutant allele.

Any one of, or combination of, the above-mentioned strategies for deactivating a mutant allele may be used in the context of the invention.

Additional strategies may be used to deactivate a mutated allele. For example, in embodiments of the present invention, an RNA molecule is used to direct a CRISPR nuclease to an exon or a splice site of a mutated allele in order to create a double-stranded break (DSB), leading to insertion or deletion of nucleotides by an error-prone non-homologous end-joining (NHEJ) mechanism and formation of a frameshift mutation in the mutated allele. The frameshift mutation may result in: (1) inactivation or knockout of the mutated allele by generation of an early stop codon in the mutated allele, resulting in generation of a truncated protein; or (2) nonsense mediated mRNA decay of the transcript of the mutant allele. In further embodiments, one RNA molecule is used to direct a CRISPR nuclease to a promotor of a mutated allele.

In some embodiments, the method of deactivating a mutated allele further comprises enhancing activity of the functional protein such as by providing a protein/peptide, a nucleic acid encoding a protein/peptide, or a small molecule such as a chemical compound, capable of activating/enhancing activity of the functional protein.

According to some embodiments, the present disclosure provides an RNA sequence (‘RNA molecule’) which binds to/associates with and/or directs the RNA guided DNA nuclease e.g., CRISPR nuclease to a sequence comprising at least one nucleotide which differs between a mutated allele and a functional allele (e.g., SNP) of a gene of interest (i.e., a sequence of the mutated allele which is not present in the functional allele).

In some embodiments, the method comprises the steps of: contacting a mutated allele of a gene of interest with an allele-specific RNA molecule and a CRISPR nuclease e.g., a Cas9 protein, wherein the allele-specific RNA molecule and the CRISPR nuclease e.g., Cas9 associate with a nucleotide sequence of the mutated allele of the gene of interest which differs by at least one nucleotide from a nucleotide sequence of a functional allele of the gene of interest, thereby modifying or knocking-out the mutated allele.

In some embodiments, the allele-specific RNA molecule and a CRISPR nuclease is introduced to a cell encoding the gene of interest. In some embodiments, the cell encoding the gene of interest is in a mammalian subject. In some embodiments, the cell encoding the gene of interest is in a plant.

In some embodiments, the cleaved mutated allele is further subjected to insertion or deletion (indel) by an error prone non-homologous end joining (NHEJ) mechanism, generating a frameshift in the mutated allele's sequence. In some embodiments, the generated frameshift results in inactivation or knockout of the mutated allele. In some embodiments, the generated frameshift creates an early stop codon in the mutated allele and results in generation of a truncated protein. In such embodiments, the method results in the generation of a truncated protein encoded by the mutated allele and a functional protein encoded by the functional allele. In some embodiments, a frameshift generated in a mutated allele using the methods of the invention results in nonsense-mediated mRNA decay of the transcript of the mutant allele.

In some embodiments, the mutated allele is an allele of the BEST1 gene. In some embodiments, the RNA molecule targets a SNP which co-exists with/is genetically linked to the mutated sequence associated with Best Vitelliform Macular Dystrophy genetic disorder. In some embodiments, the RNA molecule targets a SNP which is highly prevalent in the population and exists in the mutated allele having the mutated sequence associated with Best Vitelliform Macular Dystrophy genetic disorder and not in the functional allele of an individual subject to be treated. In some embodiments, a disease-causing mutation within a mutated BEST1 allele is targeted.

In some embodiments, the SNP is within an exon of the gene of interest. In such embodiments, a guide sequence portion of an RNA molecule may be designed to associate with a sequence of the exon of the gene of interest.

In some embodiments, SNP is within an intron or an exon of the gene of interest. In some embodiments, SNP is in close proximity to a splice site between the intron and the exon. In some embodiments, the close proximity to a splice site is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream or downstream to the splice site. Each possibility represents a separate embodiment of the present invention. In such embodiments, a guide sequence portion of an RNA molecule may be designed to associate with a sequence of the gene of interest which comprises the splice site.

In some embodiments, the method is utilized for treating a subject having a disease phenotype resulting from the heterozygote BEST1 gene. In such embodiments, the method results in improvement, amelioration or prevention of the disease phenotype.

Embodiments referred to above refer to a CRISPR nuclease, RNA molecule(s), and tracrRNA being effective in a subject or cells at the same time. The CRISPR, RNA molecule(s), and tracrRNA can be delivered substantially at the same time or can be delivered at different times but have effect at the same time. For example, this includes delivering the CRISPR nuclease to the subject or cells before the RNA molecule and/or tracr RNA is substantially extant in the subject or cells.

In some embodiments, the cell is a retinal cell. In some embodiments, the cell is a Retinal pigment epithelium (RPE) cell.

Dominant Genetic Disorders

One of skill in the art will appreciate that all subjects with any type of heterozygote genetic disorder (e.g., dominant genetic disorder) may be subjected to the methods described herein. In one embodiment, the present invention may be used to target a gene involved in, associated with, or causative of dominant genetic disorders such as, for example treating Best Vitelliform Macular Dystrophy. In some embodiments, the dominant genetic disorder is treating Best Vitelliform Macular Dystrophy. In some embodiments, the target gene is the BEST1 gene (Entrez Gene, gene ID No: 7439).

CRISPR Nucleases and PAM Recognition

In some embodiments, the sequence specific nuclease is selected from CRISPR nucleases, or a functional variant thereof. In some embodiments, the sequence specific nuclease is an RNA guided DNA nuclease. In such embodiments, the RNA sequence which guides the RNA guided DNA nuclease (e.g., Cpf1) binds to and/or directs the RNA guided DNA nuclease to the sequence comprising at least one nucleotide which differs between a mutated allele and its counterpart functional allele (e.g., SNP). In some embodiments, the CRISPR complex does not further comprise a tracrRNA. In a non-limiting example, in which the RNA guided DNA nuclease is a CRISPR protein, the at least one nucleotide which differs between the dominant mutated allele and the functional allele may be within the PAM site and/or proximal to the PAM site within the region that the RNA molecule is designed to hybridize to. A skilled artisan will appreciate that RNA molecules can be engineered to bind to a target of choice in a genome by commonly known methods in the art.

In embodiments of the present invention, a type II CRISPR system utilizes a mature crRNA:tracrRNA complex directs a CRISPR nuclease, e.g. Cas9, to the target DNA via Watson-Crick base-pairing between the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. The CRISPR nuclease then mediates cleavage of target DNA to create a double-stranded break within the protospacer. A skilled artisan will appreciate that each of the engineered RNA molecule of the present invention is further designed such as to associate with a target genomic DNA sequence of interest next to a protospacer adjacent motif (PAM), e.g., a PAM matching the sequence relevant for the type of CRISPR nuclease utilized, such as for a non-limiting example, NGG or NAG, wherein “N” is any nucleobase, for Streptococcus pyogenes Cas9 WT (SpCAS9); NNGRRT for Staphylococcus aureus (SaCas9); NNNVRYM for Jejuni Cas9 WT; NGAN or NGNG for SpCas9-VQR variant; NGCG for SpCas9-VRER variant; NGAG for SpCas9-EQR variant; NNNNGATT for Neisseria meningitidis (NmCas9); or TTTV for Cpf1. RNA molecules of the present invention are each designed to form complexes in conjunction with one or more different CRISPR nucleases and designed to target polynucleotide sequences of interest utilizing one or more different PAM sequences respective to the CRISPR nuclease utilized.

In some embodiments, an RNA-guided DNA nuclease e.g., a CRISPR nuclease, may be used to cause a DNA break at a desired location in the genome of a cell. The most commonly used RNA-guided DNA nucleases are derived from CRISPR systems, however, other RNA-guided DNA nucleases are also contemplated for use in the genome editing compositions and methods described herein. For instance, see U.S. Patent Publication No. 2015-0211023, incorporated herein by reference.

CRISPR systems that may be used in the practice of the invention vary greatly. CRISPR systems can be a type I, a type II, or a type III system. Non-limiting examples of suitable CRISPR proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Casl Od, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cul966.

In some embodiments, the RNA-guided DNA nuclease is a CRISPR nuclease derived from a type II CRISPR system (e.g., Cas9). The CRISPR nuclease may be derived from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Neisseria meningitidis, Treponema denticola, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium Finegoldia magna, Natranaerobius thermophilus, Pelotomaculumthermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, or any species which encodes a CRISPR nuclease with a known PAM sequence. CRISPR nucleases encoded by uncultured bacteria may also be used in the context of the invention. (See Burstein et al. Nature, 2017). Variants of CRIPSR proteins having known PAM sequences e.g., spCas9 D1135E variant, spCas9 VQR variant, spCas9 EQR variant, or spCas9 VRER variant may also be used in the context of the invention.

Thus, an RNA guided DNA nuclease of a CRISPR system, such as a Cas9 protein or modified Cas9 or homolog or ortholog of Cas9, or other RNA guided DNA nucleases belonging to other types of CRISPR systems, such as Cpf1 and its homologs and orthologs, may be used in the compositions of the present invention.

In certain embodiments, the CRIPSR nuclease may be a “functional derivative” of a naturally occurring Cas protein. A “functional derivative” of a native sequence polypeptide is a compound having a qualitative biological property in common with a native sequence polypeptide. “Functional derivatives” include, but are not limited to, fragments of a native sequence and derivatives of a native sequence polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence polypeptide. A biological activity contemplated herein is the ability of the functional derivative to hydrolyze a DNA substrate into fragments. The term “derivative” encompasses both amino acid sequence variants of polypeptide, covalent modifications, and fusions thereof. Suitable derivatives of a Cas polypeptide or a fragment thereof include but are not limited to mutants, fusions, covalent modifications of Cas protein or a fragment thereof. Cas protein, which includes Cas protein or a fragment thereof, as well as derivatives of Cas protein or a fragment thereof, may be obtainable from a cell or synthesized chemically or by a combination of these two procedures. The cell may be a cell that naturally produces Cas protein, or a cell that naturally produces Cas protein and is genetically engineered to produce the endogenous Cas protein at a higher expression level or to produce a Cas protein from an exogenously introduced nucleic acid, which nucleic acid encodes a Cas that is same or different from the endogenous Cas. In some cases, the cell does not naturally produce Cas protein and is genetically engineered to produce a Cas protein.

In some embodiments, the CRISPR nuclease is Cpf1. Cpf1 is a single RNA-guided endonuclease which utilizes a T-rich protospacer-adjacent motif. Cpf1 cleaves DNA via a staggered DNA double-stranded break. Two Cpf1 enzymes from Acidaminococcus and Lachnospiraceae have been shown to carry out efficient genome-editing activity in human cells. (See Zetsche et al. (2015) Cell).

Thus, an RNA guided DNA nuclease of a Type II CRISPR System, such as a Cas9 protein or modified Cas9 or homologs, orthologues, or variants of Cas9, or other RNA guided DNA nucleases belonging to other types of CRISPR systems, such as Cpf1 and its homologs, orthologues, or variants, may be used in the present invention.

In some embodiments, the guide molecule comprises one or more chemical modifications which imparts a new or improved property (e.g., improved stability from degradation, improved hybridization energetics, or improved binding properties with an RNA guided DNA nuclease). Suitable chemical modifications include, but are not limited to: modified bases, modified sugar moieties, or modified inter-nucleoside linkages. Non-limiting examples of suitable chemical modifications include: 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 2′-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2′-O-methylpseudouridine, “beta. D-galactosylqueuosine”, 2′-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methvladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, “2,2-dimethylguanosine”, 2-methyladenosine, 2-methylguanosine, 3-methylcytidine, 5-methylcytidine, N6-methyladenosine, 7-methylguanosine, 5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine, “beta, D-mannosylqueuosine”, 5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N-((9-beta-D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine, N-((9-beta-D-ribofuranosylpurine-6-yl)N-methylcarbamoyl)threonine, uridine-5-oxyacetic acid-methylester, uridine-5-oxyacetic acid, wybutoxosine, queuosine, 2-thiocytidine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine. N-((9-beta-D-ribofuranosylpurine-6-yl)-carbamoyl)threonine, 2′-O-methyl-5-methyluridine, 2′-O-methyluridine, wybutosine, “3-(3-amino-3-carboxy-propyl)uridine, (acp3)u”, 2′-O-methyl (M), 3′-phosphorothioate (MS), 3′-thioPACE (MSP), pseudouridine, or 1-methyl pseudo-uridine. Each possibility represents a separate embodiment of the present invention.

Guide Sequences which Specifically Target a Mutant Allele

A given gene may contain thousands of SNPs. Utilizing a 24 base pair target window for targeting each SNP in a gene would require hundreds of thousands of guide sequences. Any given guide sequence when utilized to target a SNP may result in degradation of the guide sequence, limited activity, no activity, or off-target effects. Accordingly, suitable guide sequences are necessary for targeting a given gene. By the present invention, a novel set of guide sequences have been identified for knocking out expression of a mutated BEST1 protein, inactivating a mutant BEST1 gene allele, and treating Best Vitelliform Macular Dystrophy.

The present disclosure provides guide sequences capable of specifically targeting a mutated allele for inactivation while leaving the functional allele unmodified. The guide sequences of the present invention are designed to, and are most likely to, specifically differentiate between a mutated allele and a functional allele. Of all possible guide sequences which target a mutated allele desired to be inactivated, the specific guide sequences disclosed herein are specifically effective to function with the disclosed embodiments.

Briefly, the guide sequences may have properties as follows: (1) target SNP/insertion/deletion/indel with a high prevalence in the general population, in a specific ethnic population or in a patient population is above 1% and the SNP/insertion/deletion/indel heterozygosity rate in the same population is above 1%; (2) target a location of a SNP/insertion/deletion/indel proximal to a portion of the gene e.g., within 5 k bases of any portion of the gene, for example, a promoter, a UTR, an exon or an intron; and (3) target a mutant allele using an RNA molecule which targets a founder or common pathogenic mutations for the disease/gene. In some embodiments, the prevalence of the SNP/insertion/deletion/indel in the general population, in a specific ethnic population or in a patient population is above 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% and the SNP/insertion/deletion/indel heterozygosity rate in the same population is above 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. Each possibility represents a separate embodiment and may be combined at will.

For each gene, according to SNP/insertion/deletion/indel any one of the following strategies may be used to deactivate the mutated allele: (1) Knockout strategy using one RNA molecule—one RNA molecule is utilized to direct a CRISPR nuclease to a mutated allele and create a double-strand break (DSB) leading to formation of a frameshift mutation in an exon or in a splice site region of the mutated allele; (2) Knockout strategy using two RNA molecules—two RNA molecules are utilized. A first RNA molecule targets a region in the promoter or an upstream region of a mutated allele and another RNA molecule targets downstream of the first RNA molecule in a promoter, exon, or intron of the mutated allele; (3) Exon(s) skipping strategy—one RNA molecule may be used to target a CRISPR nuclease to a splice site region, either at the 5′ end of an intron (donor sequence) or the 3′ end of an intron (acceptor sequence), in order to destroy the splice site. Alternatively, two RNA molecules may be utilized such that a first RNA molecule targets an upstream region of an exon and a second RNA molecule targets a region downstream of the first RNA molecule, thereby excising the exon(s). Based on the locations of identified SNPs/insertions/deletions/indels for each mutant allele, any one of, or a combination of, the above-mentioned methods to deactivate the mutant allele may be utilized.

When only one RNA molecule is used is that the location of the SNP is in an exon or in close proximity (e.g., within 20 basepairs) to a splice site between the intron and the exon. When two RNA molecules are used, guide sequences may target two SNPs such that the first SNP is upstream of exon 1 e.g., within the 5′ untranslated region, or within the promoter or within the first 2 kilobases 5′ of the transcription start site, and the second SNP is downstream of the first SNP e.g., within the first 2 kilobases 5′ of the transcription start site, or within intron 1, 2 or 3, or within exon 1, exon 2, or exon 3.

Guide sequences of the present invention may target a SNP in the upstream portion of the targeted gene, preferably upstream of the last exon of the targeted gene. Guide sequences may target a SNP upstream to exon 1, for example within the 5′ untranslated region, or within the promoter or within the first 4-5 kilobases 5′ of the transcription start site.

Guide sequences of the present invention may also target a SNP within close proximity (e.g., within 50 basepairs, more preferably with 20 basepairs) to a known protospacer adjacent motif (PAM) site.

Guide sequences of the present invention also may target: (1) a heterozygous SNP for the targeted gene; (2) a heterozygous SNPs upstream and downstream of the gene; (3) a SNPs with a prevalence of the SNP/insertion/deletion/indel in the general population, in a specific ethnic population, or in a patient population above 1%; (4) have a guanine-cytosine content of greater than 30% and less than 85%; (5) have no repeat of 4 or more thymine/uracil or 8 or more guanine, cytosine, or adenine; (6) having no off-target identified by off-target analysis; and (7) preferably target Exons over Introns or be upstream of a SNP rather than downstream of a SNP.

In embodiments of the present invention, the SNP may be upstream or downstream of the gene. In embodiments of the present invention, the SNP is within 4,000 base pairs upstream or downstream of the gene.

The at least one nucleotide which differs between the mutated allele and the functional allele, may be upstream, downstream or within the sequence of the disease-causing mutation of the gene of interest. The at least one nucleotide which differs between the mutated allele and the functional allele, may be within an exon or within an intron of the gene of interest. In some embodiments, the at least one nucleotide which differs between the mutated allele and the functional allele is within an exon of the gene of interest. In some embodiments, the at least one nucleotide which differs between the mutated allele and the functional allele is within an intron or an exon of the gene of interest, in close proximity to a splice site between the intron and the exon e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream or downstream to the splice site.

In some embodiments, the at least one nucleotide is a single nucleotide polymorphisms (SNPs). In some embodiments, each of the nucleotide variants of the SNP may be expressed in the mutated allele. In some embodiments, the SNP may be a founder or common pathogenic mutation.

Guide sequences may target a SNP which has both (1) a high prevalence in the general population e.g., above 1% in the population; and (2) a high heterozygosity rate in the population, e.g., above 1%. Guide sequences may target a SNP that is globally distributed. A SNP may be a founder or common pathogenic mutation. In some embodiments, the prevalence in the general population is above 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. Each possibility represents a separate embodiment. In some embodiments, the heterozygosity rate in the population is above 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. Each possibility represents a separate embodiment.

In some embodiments, the at least one nucleotide which differs between the mutated allele and the functional allele is linked to/co-exists with the disease-causing mutation in high prevalence in a population. In such embodiments, “high prevalence” refers to at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Each possibility represents a separate embodiment of the present invention. In one embodiment, the at least one nucleotide which differs between the mutated allele and the functional allele, is a disease-associated mutation. In some embodiments, the SNP is highly prevalent in the population. In such embodiments, “highly prevalent” refers to at least 10%, 11%, 12%, 13%, 14%, 15%, 20%, 30%, 40%, 50%, 60%, or 70% of a population. Each possibility represents a separate embodiment of the present invention.

Guide sequences of the present invention may satisfy any one of the above criteria and are most likely to differentiate between a mutated allele from its corresponding functional allele.

In some embodiments the RNA molecule targets a SNP/WT sequence linked to SNPs as shown in Table 1 below. The SNP details are indicated in the 1^(st) column and include: SNP ID No. (based on NCBI's 2018 database of Single Nucleotide Polymorphisms (dbSNP)). For variants with no available rs number variants characteristic are indicated based on gnomAD 2018 browser database. The 2^(nd) column indicates an assigned identifier for each SNP. The 3^(rd) column indicates the location of each SNP on the BEST1 gene.

TABLE 1 BEST1 gene SNPs SNP SNP location in the RSID No. gene rs1800009  s1 Exon_10 of 11 rs2524294  s2 Intron_2 of 10 rs909268  s3 Intron_4 of 10 rs2668898  s4 Intron_6 of 10 rs972355  s5 upstream −18 bp rs972353  s6 Exon_1 of 11 rs2736597  s7 Intron_1 of 10 rs1800007  s8 Exon_2 of 11 rs760306  s9 Intron_4 of 10 rs974121 s10 upstream −1048 bp rs168991 s11 Intron_2 of 10 rs195161 s12 Intron_5 of 10 rs149698 s13 Exon_10 of 11 rs1534842 s14 upstream −3765 bp rs3758976 s15 upstream −250 bp rs1800008 s16 Exon_10 of 11 rs195158 s17 Intron_7 of 10 rs195157 s18 Intron_9 of 10 rs195156 s19 Intron_9 of 10 rs2009875 s20 upstream −3323 bp rs2955684 s21 Intron_6 of 10 rs2955683 s22 Intron_6 of 10 rs17185413 s23 Intron_10 of 10 rs972354 s24 Exon_1 of 11 rs195163 s25 Intron_4 of 10 rs2668897 s26 Intron_10 of 10 rs1109748 s27 Exon_3 of 11 rs195160 s28 Intron_7 of 10 rs183176 s29 Intron_2 of 10 rs195167 s30 Intron_2 of 10 rs195165 s31 Intron_3 of 10 rs195164 s32 Intron_4 of 10 rs2736594 s33 Intron_2 of 10 rs195162 s34 Intron_5 of 10 rs113492158 s35 Intron_6 of 10 rs195166 s36 Intron_2 of 10 rs741886 s37 Intron_5 of 10 rs2736596 s38 Intron_1 of 10 rs1801621 s39 Exon_11 of 11 rs17156609 s40 downstream +42 bp rs1534843 s41 upstream −3860 bp rs73491300 s42 upstream −224 bp rs74754540 s43 upstream −998 bp rs112769638 s44 Intron_1 of 10 rs71471844 s45 Intron_7 of 10 rs1801393 s46 Exon_3 of 11 rs185387478 s47 Intron_6 of 10 rs180929734 s48 Intron_6 of 10 rs111352087 s49 Intron_2 of 10 rs57890952 s50 Intron_2 of 10 rs145834822 s51 Intron_9 of 10 rs1801390 s52 Exon_9 of 11 rs17156602 s53 Intron_9 of 10 rs11825719 s54 Intron_1 of 10 rs75281081 s55 Exon_11 of 11 rs57815521 s56 Intron_9 of 10 rs73493223 s57 Intron_9 of 10 rs111677305 s58 Intron_9 of 10 rs74746022 s59 Intron_2 of 10 rs75769763 s60 Intron_5 of 10 rs114450910 s61 Intron_4 of 10 rs74369809 s62 Intron_9 of 10 rs78054615 s63 Intron_2 of 10 rs144630276 s64 Intron_2 of 10 rs141507235 s65 Intron_6 of 10 rs114944671 s66 Intron_3 of 10 rs1801327 s67 Exon_11 of 11 rs78012644 s68 Intron_7 of 10 rs112665957 s69 Intron_2 of 10 rs139745332 s70 Intron_2 of 10 rs77543508 s71 Intron_2 of 10 rs78545127 s72 upstream −2638 bp rs116516743 s73 upstream −2116 bp rs1805140 s74 Exon_6 of 11 rs73493205 s75 Intron_1 of 10 rs77651946 s76 Intron_1 of 10 rs195159 s77 Intron_7 of 10 rs195155 s78 Intron_10 of 10 rs2727272 s79 Intron_2 of 10 rs2668899 s80 Intron_2 of 10 rs2736595 s81 Intron_2 of 10 rs56215258 s82 Intron_10 of 10 rs174481 s83 upstream −960 bp rs168990 s84 Intron_4 of 10 rs111509315 s85 Intron_9 of 10 rs1735379 s86 Intron_7 of 10 rs112720784 s87 Intron_2 of 10 Delivery to Cells

The RNA molecule compositions described herein may be delivered to a target cell by any suitable means. RNA molecule compositions of the present invention may be targeted to any cell which contains and/or expresses a dominant negative allele, including any mammalian or plant cell. For example, in one embodiment a guide sequence specifically targets a mutated BEST1 allele and the target cell is a retinal cell such as pigment epithelium (RPE), photoreceptors (e.g., rod and cone), glial cells (e.g., Müller), and ganglion cells. In some embodiments, the target cell is RPE. Further, the nucleic acid compositions described herein may be delivered as DNA molecules, RNA molecules, Ribonucleoproteins (RNP), nucleic acid vectors, or any combination thereof.

In some embodiments, the RNA molecule comprises a chemical modification. Non-limiting examples of suitable chemical modifications include 2′-0-methyl (M), 2′-0-methyl, 3′phosphorothioate (MS) or 2′-0-methyl, 3′thioPACE (MSP), pseudouridine, and 1-methyl pseudo-uridine. Each possibility represents a separate embodiment of the present invention.

Any suitable viral vector system may be used to deliver nucleic acid compositions e.g., the RNA molecule compositions of the subject invention. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids and target tissues. In certain embodiments, nucleic acids are administered for in vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. For a review of gene therapy procedures, see Anderson (1992) Science 256:808-813; Nabel & Felgner (1993) TIBTECH 11:211-217; Mitani & Caskey (1993) TIBTECH 11:162-166; Dillon (1993) TIBTECH 11:167-175; Miller (1992) Nature 357:455-460; Van Brunt (1988) Biotechnology 6(10):1149-1154; Vigne (1995) Restorative Neurology and Neuroscience 8:35-36; Kremer & Perricaudet (1995) British Medical Bulletin 51(1):31-44; Haddada et al. (1995) in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds.); and Yu et al. (1994) Gene Therapy 1:13-26.

Methods of non-viral delivery of nucleic acids and/or proteins include electroporation, lipofection, microinjection, biolistics, particle gun acceleration, virosomes, liposomes, immunoliposomes, lipid nanoparticles (LNPs), polycation or lipid:nucleic acid conjugates, artificial virions, and agent-enhanced uptake of nucleic acids or can be delivered to plant cells by bacteria or viruses (e.g., Agrobacterium, Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, tobacco mosaic virus, potato virus X, cauliflower mosaic virus and cassava vein mosaic virus). (See, e.g., Chung et al. (2006) Trends Plant Sci. 11(1):1-4). Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar), can also be used for delivery of nucleic acids. Cationic-lipid mediated delivery of proteins and/or nucleic acids is also contemplated as an in vivo or in vitro delivery method. (See Zuris et al. (2015) Nat. Biotechnol. 33(1):73-80; see also Coelho et al. (2013) N. Engl. J. Med. 369, 819-829; Judge et al. (2006) Mol. Ther. 13, 494-505; and Basha et al. (2011) Mol. Ther. 19, 2186-2200).

Additional exemplary nucleic acid delivery systems include those provided by Amaxa® Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc., (see, e.g., U.S. Pat. No. 6,008,336). Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents are sold commercially (e.g., Transfectam™, Lipofectin™ and Lipofectamine™ RNAiMAX). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (See, e.g., Crystal (1995) Science 270:404-410; Blaese et al. (1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994) Bioconjugate Chem. 5:382-389; Remy et al. (1994) Bioconjugate Chem. 5:647-654; Gao et al. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992) Cancer Res. 52:4817-4820; U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleic acids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis. Once in the cell, the contents are released (See MacDiarmid et al (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for viral mediated delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of nucleic acids include, but are not limited to, retroviral, lentivirus, adenoviral, adeno-associated, vaccinia and herpes simplex virus vectors for gene transfer.

The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system depends on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (See, e.g., Buchschacher et al. (1992) J. Virol. 66:2731-2739; Johann et al. (1992) J. Virol. 66:1635-1640; Sommerfelt et al. (1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al. (1991) J. Virol. 65:2220-2224; PCT/US94/05700).

At least six viral vector approaches are currently available for gene transfer in clinical trials, which utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been used in clinical trials (Dunbar et al. (1995) Blood 85:3048-305; Kohn et al. (1995) Nat. Med. 1:1017-102; Malech et al. (1997) PNAS 94:22 12133-12138). PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al. (1995). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al. (1997) Immunol Immunother. 44(1):10-20; Dranoff et al. (1997) Hum. Gene Ther. 1:111-2).

Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, AAV, and Psi-2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additionally, AAV can be produced at clinical scale using baculovirus systems (see U.S. Pat. No. 7,479,554).

In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. Accordingly, a viral vector can be modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the outer surface of the virus. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al. (1995) Proc. Natl. Acad. Sci. USA 92:9747-9751, reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other virus-target cell pairs, in which the target cell expresses a receptor and the virus expresses a fusion protein comprising a ligand for the cell-surface receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences which favor uptake by specific target cells.

Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravitreal, intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with a nucleic acid composition, and re-infused back into the subject organism (e.g., patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art (See, e.g., Freshney et al. (1994) Culture of Animal Cells, A Manual of Basic Technique, 3rd ed, and the references cited therein for a discussion of how to isolate and culture cells from patients).

Suitable cells include, but are not limited to, eukaryotic cells and/or cell lines. Non-limiting examples of such cells or cell lines generated from such cells include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6 cells, any plant cell (differentiated or undifferentiated), as well as insect cells such as Spodopterafugiperda (Sf), or fungal cells such as Saccharomyces, Pichia and Schizosaccharomyces. In certain embodiments, the cell line is a CHO-K1, MDCK or HEK293 cell line. Additionally, primary cells may be isolated and used ex vivo for reintroduction into the subject to be treated following treatment with a guided nuclease system (e.g. CRISPR/Cas). Suitable primary cells include peripheral blood mononuclear cells (PBMC), and other blood cell subsets such as, but not limited to, CD4+ T cells or CD8+ T cells. Suitable cells also include stem cells such as, by way of example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells (CD34+), neuronal stem cells and mesenchymal stem cells.

In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow. Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN-gamma, and TNF-alpha are known (as a non-limiting example see, Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+(T cells), CD45+(panB cells), GR-1 (granulocytes), and Tad (differentiated antigen presenting cells) (as a non-limiting example see Inaba et al. (1992) J. Exp. Med. 176:1693-1702). Stem cells that have been modified may also be used in some embodiments.

Any one of the RNA molecule compositions described herein is suitable for genome editing in post-mitotic cells or any cell which is not actively dividing, e.g., arrested cells. Examples of post-mitotic cells which may be edited using a composition of the present invention include, but are not limited to, a photoreceptor cell, a rod photoreceptor cell, a cone photoreceptor cell, a retinal pigment epithelium (RPE), a glial cell, Müller cell, and a ganglion.

Vectors (e.g., retroviruses, liposomes, etc.) containing therapeutic nucleic acid compositions can also be administered directly to an organism for transduction of cells in vivo. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application (e.g., eye drops and cream) and electroporation. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route. According to some embodiments, the composition is delivered via sub-retinal injection. According to some embodiments, the composition is delivered via intravitreal injection.

Vectors suitable for introduction of transgenes into immune cells (e.g., T-cells) include non-integrating lentivirus vectors. See, e.g., U.S. Patent Publication No. 2009-0117617.

Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions available, as described below (See, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

In accordance with some embodiments, there is provided an RNA molecule which binds to/associates with and/or directs the RNA guided DNA nuclease to a sequence comprising at least one nucleotide which differs between a mutated allele and a functional allele (e.g., SNP) of a gene of interest (i.e., a sequence of the mutated allele which is not present in the functional allele). The sequence may be within the disease associated mutation. The sequence may be upstream or downstream to the disease associated mutation. Any sequence difference between the mutated allele and the functional allele may be targeted by an RNA molecule of the present invention to inactivate the mutant allele, or otherwise disable its dominant disease-causing effects, while preserving the activity of the functional allele.

The disclosed compositions and methods may also be used in the manufacture of a medicament for treating dominant genetic disorders in a patient.

Examples of RNA Guide Sequences which Specifically Target Mutated Alleles of BEST1 Gene

Although a large number of guide sequences can be designed to target a mutated allele, the nucleotide sequences described in Tables 2 identified by SEQ ID NOs: 1-3010 below were specifically selected to effectively implement the methods set forth herein and to effectively discriminate between alleles.

Referring to columns 1-4, each of SEQ ID NOs. 1-3010 indicated in column 1 corresponds to an engineered guide sequence. The corresponding SNP details are indicated in column 2. The SNP details indicated in the 2nd column include the assigned identifier for each SNP corresponding to a SNP ID indicated in Table 1. Column 3 indicates whether the target of each guide sequence is the BEST1 gene polymorph or wild type (REF) sequence. Column 4 indicates the guanine-cytosine content of each guide sequence.

Table 2 shows guide sequences designed for use as described in the embodiments above to associate with different SNPs within a sequence of a mutated BEST1 allele. Each engineered guide molecule is further designed such as to associate with a target genomic DNA sequence of interest that lies next to a protospacer adjacent motif (PAM), e.g., a PAM matching the sequence NGG or NAG, where “N” is any nucleobase. The guide sequences were designed to work in conjunction with one or more different CRISPR nucleases, including, but not limited to, e.g. SpCas9WT (PAM SEQ: NGG), SpCas9.VQR.1 (PAM SEQ: NGAN), SpCas9.VQR.2 (PAM SEQ: NGNG), SpCas9.EQR (PAM SEQ: NGAG), SpCas9.VRER (PAM SEQ: NGCG), SaCas9WT (PAM SEQ: NNGRRT), NmCas9WT (PAM SEQ: NNNNGATT), Cpf1 (PAM SEQ: TTTV), or JeCas9WT (PAM SEQ: NNNVRYM). RNA molecules of the present invention are each designed to form complexes in conjunction with one or more different CRISPR nucleases and designed to target polynucleotide sequences of interest utilizing one or more different PAM sequences respective to the CRISPR nuclease utilized

TABLE 2 SEQ ID SNP ID Target SEQ ID SNP ID Target NO: (Table 1) (SNP/REF) % GC NO: (Table 1) (SNP/REF) % GC 1 s1 BOTH 45% 4 s5 BOTH 55% 2 s1 BOTH 45% 5 s5 BOTH 60% 3 s2 BOTH 70% 6 s6 BOTH 45% 7 s6 BOTH 50% 43 s44 BOTH 30% 8 s6 BOTH 50% 44 s45 BOTH 50% 9 s8 BOTH 45% 45 s45 BOTH 50% 10 s9 BOTH 50% 46 s45 BOTH 50% 11 s9 BOTH 55% 47 s45 BOTH 50% 12 s12 BOTH 65% 48 s46, s27 BOTH, REF 50% 13 s13 BOTH 60% 49 s46, s27 REF, REF 50% 14 s14 BOTH 60% 50 s48, s47 REF, BOTH 65% 15 s14 BOTH 65% 51 s48, s47 BOTH, BOTH 60% 16 s15 BOTH 75% 52 s48, s47 REF, REF 65% 17 s16 BOTH 40% 53 s48, s47 REF, REF 65% 18 s16 BOTH 40% 54 s48, s47 REF, REF 65% 19 s17 BOTH 55% 55 s48, s47 REF, REF 70% 20 s20 BOTH 45% 56 s48, s47 REF, REF 65% 21 s21, s22 BOTH, BOTH 45% 57 s48 BOTH 55% 22 s23 BOTH 50% 58 s51 BOTH 45% 23 s23 BOTH 50% 59 s52 BOTH 45% 24 s24 BOTH 50% 60 s52 BOTH 65% 25 s24 BOTH 50% 61 s54 BOTH 55% 26 s27 BOTH 50% 62 s54 BOTH 40% 27 s29 BOTH 35% 63 s54 BOTH 45% 28 s31 BOTH 65% 64 s55 BOTH 45% 29 s31 BOTH 65% 65 s55 BOTH 45% 30 s32 BOTH 45% 66 s56 BOTH 30% 31 s32 BOTH 45% 67 s57 BOTH 50% 32 s35 BOTH 55% 68 s57 BOTH 50% 33 s35 BOTH 55% 69 s58 BOTH 40% 34 s36 BOTH 40% 70 s58 BOTH 45% 35 s38 BOTH 60% 71 s59 BOTH 50% 36 s40 BOTH 45% 72 s60 BOTH 55% 37 s41 BOTH 55% 73 s60 BOTH 50% 38 s41 BOTH 70% 74 s61 BOTH 55% 39 s41 BOTH 55% 75 s61 BOTH 75% 40 s42 BOTH 45% 76 s61 BOTH 55% 41 s43 BOTH 40% 77 s62 BOTH 40% 42 s43 BOTH 50% 78 s62 BOTH 55% 79 s62 BOTH 40% 115 s3 SNP 60% 80 s63 BOTH 45% 116 s3 SNP 55% 81 s63 BOTH 55% 117 s3 REF 55% 82 s63 BOTH 35% 118 s3 SNP 55% 83 s65 BOTH 50% 119 s3 REF 55% 84 s65 BOTH 45% 120 s3 REF 50% 85 s66 BOTH 80% 121 s3 SNP 50% 86 s66 BOTH 80% 122 s4 REF 40% 87 s67 BOTH 35% 123 s4 SNP 45% 88 s69 BOTH 65% 124 s4 REF 45% 89 s69 BOTH 60% 125 s4 SNP 50% 90 s69 BOTH 70% 126 s4 SNP 50% 91 s71 BOTH 70% 127 s4 REF 45% 92 s71 BOTH 70% 128 s4 SNP 40% 93 s72 BOTH 40% 129 s4 REF 35% 94 s72 BOTH 45% 130 s5 SNP 65% 95 s73 BOTH 60% 131 s5 REF 65% 96 s74 BOTH 50% 132 s5 REF 65% 97 s74 BOTH 50% 133 s5 SNP 65% 98 s76 BOTH 45% 134 s5 SNP 70% 99 s76 BOTH 40% 135 s5 REF 70% 100 s79 REF 35% 136 s5 REF 65% 101 s80 SNP 45% 137 s5 SNP 65% 102 s1 SNP 45% 138 s5 REF 65% 103 s1 REF 40% 139 s5 SNP 65% 104 s1 SNP 50% 140 s6 SNP 45% 105 s1 REF 45% 141 s6 REF 40% 106 s1 SNP 45% 142 s6 SNP 45% 107 s1 REF 40% 143 s6 SNP 45% 108 s1 SNP 50% 144 s6 REF 40% 109 s1 REF 45% 145 s6 REF 40% 110 s2 SNP 65% 146 s7 REF 55% 111 s2 REF 70% 147 s7 SNP 50% 112 s2 SNP 55% 148 s7 REF 45% 113 s2 REF 60% 149 s7 SNP 40% 114 s3 REF 60% 150 s7 REF 60% 151 s7 SNP 55% 187 s12 SNP 70% 152 s7 SNP 50% 188 s12 REF 65% 153 s7 REF 55% 189 s12 REF 65% 154 s7 REF 65% 190 s12 REF 70% 155 s7 REF 55% 191 s12 SNP 75% 156 s7 SNP 50% 192 s12 SNP 70% 157 s7 SNP 60% 193 s12 SNP 70% 158 s7 SNP 50% 194 s12 REF 65% 159 s7 REF 55% 195 s12 REF 65% 160 s7 SNP 50% 196 s12 SNP 70% 161 s7 REF 55% 197 s12 SNP 65% 162 s7 REF 60% 198 s12 REF 60% 163 s7 SNP 55% 199 s13 SNP 65% 164 s7 REF 55% 200 s13 REF 70% 165 s7 SNP 50% 201 s13 REF 70% 166 s8 SNP 45% 202 s13 SNP 65% 167 s8 REF 45% 203 s13 REF 70% 168 s8 SNP 50% 204 s13 SNP 65% 169 s8 REF 45% 205 s14 SNP 65% 170 s8 SNP 50% 206 s14 REF 60% 171 s8 REF 40% 207 s14 SNP 55% 172 s9 SNP 50% 208 s14 REF 50% 173 s9 REF 55% 209 s14 REF 50% 174 s9 SNP 65% 210 s14 REF 65% 175 s9 REF 70% 211 s14 SNP 55% 176 s9 SNP 55% 212 s14 REF 60% 177 s9 REF 60% 213 s14 SNP 70% 178 s9 SNP 60% 214 s14 REF 65% 179 s9 REF 65% 215 s14 SNP 55% 180 s10 SNP 35% 216 s14 SNP 65% 181 s10 REF 40% 217 s14 REF 60% 182 s10 REF 30% 218 s14 REF 60% 183 s10 REF 30% 219 s14 SNP 70% 184 s11 REF 30% 220 s14 REF 65% 185 s12 REF 65% 221 s82 REF 45% 186 s12 SNP 70% 222 s82 REF 55% 223 s15 SNP 85% 259 s18 SNP 50% 224 s15 REF 85% 260 s18 REF 55% 225 s15 SNP 80% 261 s18 SNP 50% 226 s15 REF 80% 262 s18 REF 55% 227 s15 SNP 70% 263 s18 SNP 50% 228 s15 REF 70% 264 s18 REF 55% 229 s15 SNP 70% 265 s18 REF 50% 230 s15 REF 70% 266 s18 SNP 45% 231 s15 SNP 75% 267 s18 SNP 50% 232 s15 REF 75% 268 s18 REF 55% 233 s15 SNP 80% 269 s19 REF 45% 234 s15 REF 80% 270 s19 SNP 40% 235 s15 SNP 65% 271 s19 REF 50% 236 s15 REF 65% 272 s19 SNP 45% 237 s16 REF 45% 273 s19 REF 45% 238 s16 SNP 40% 274 s19 SNP 40% 239 s16 REF 60% 275 s19 SNP 45% 240 s16 SNP 55% 276 s19 REF 50% 241 s16 REF 60% 277 s19 SNP 55% 242 s16 SNP 55% 278 s19 REF 60% 243 s16 SNP 55% 279 s19 SNP 40% 244 s16 REF 60% 280 s19 REF 45% 245 s17 SNP 60% 281 s22 SNP 60% 246 s17 REF 65% 282 s22 SNP 65% 247 s17 REF 65% 283 s22 SNP 65% 248 s17 SNP 60% 284 s23 REF 55% 249 s17 SNP 65% 285 s23 SNP 60% 250 s17 REF 70% 286 s23 REF 55% 251 s17 REF 55% 287 s23 SNP 60% 252 s17 SNP 50% 288 s23 SNP 60% 253 s17 REF 65% 289 s23 REF 55% 254 s17 SNP 60% 290 s23 SNP 55% 255 s17 REF 65% 291 s23 REF 50% 256 s17 SNP 60% 292 s24 SNP 50% 257 s18 REF 55% 293 s24 REF 55% 258 s18 SNP 50% 294 s24 REF 55% 295 s24 REF 55% 331 s28 REF 50% 296 s24 SNP 50% 332 s28 SNP 55% 297 s24 REF 50% 333 s28 REF 55% 298 s24 SNP 45% 334 s28 SNP 60% 299 s24 REF 55% 335 s83 SNP 50% 300 s24 SNP 50% 336 s29 REF 50% 301 s24 SNP 50% 337 s29 SNP 45% 302 s25 SNP 55% 338 s29 REF 45% 303 s25 REF 60% 339 s29 SNP 40% 304 s25 REF 55% 340 s29 SNP 45% 305 s25 REF 65% 341 s29 REF 50% 306 s25 SNP 60% 342 s29 SNP 45% 307 s25 SNP 50% 343 s29 REF 50% 308 s25 REF 50% 344 s29 SNP 40% 309 s25 SNP 45% 345 s29 REF 45% 310 s26 REF 65% 346 s30 SNP 65% 311 s26 SNP 60% 347 s30 SNP 65% 312 s26 REF 70% 348 s30 SNP 75% 313 s26 SNP 65% 349 s30 SNP 70% 314 s26 SNP 60% 350 s30 SNP 70% 315 s26 REF 65% 351 s31 SNP 35% 316 s26 SNP 35% 352 s31 SNP 40% 317 s26 REF 40% 353 s31 REF 40% 318 s26 SNP 50% 354 s31 SNP 60% 319 s26 REF 55% 355 s31 REF 60% 320 s26 SNP 60% 356 s31 REF 35% 321 s26 REF 65% 357 s84 REF 70% 322 s26 REF 65% 358 s84 SNP 60% 323 s26 SNP 60% 359 s84 REF 65% 324 s26 SNP 55% 360 s84 REF 70% 325 s26 REF 60% 361 s32 REF 45% 326 s27 REF 50% 362 s32 SNP 50% 327 s27 SNP 45% 363 s32 REF 65% 328 s27 SNP 45% 364 s32 SNP 70% 329 s28 SNP 60% 365 s32 SNP 60% 330 s28 REF 55% 366 s32 REF 55% 367 s32 REF 45% 403 s37 REF 65% 368 s32 SNP 50% 404 s37 REF 75% 369 s33 SNP 55% 405 s37 REF 65% 370 s33 REF 60% 406 s37 SNP 60% 371 s33 SNP 60% 407 s37 REF 60% 372 s33 REF 65% 408 s37 SNP 55% 373 s33 SNP 60% 409 s37 SNP 70% 374 s33 REF 65% 410 s37 REF 60% 375 s34 SNP 60% 411 s37 SNP 55% 376 s34 REF 65% 412 s85 SNP 35% 377 s34 SNP 60% 413 s38 SNP 50% 378 s34 REF 65% 414 s38 REF 55% 379 s34 REF 65% 415 s38 SNP 45% 380 s34 SNP 60% 416 s38 REF 50% 381 s35 REF 60% 417 s38 REF 55% 382 s35 REF 65% 418 s38 SNP 50% 383 s35 REF 65% 419 s38 REF 50% 384 s35 SNP 60% 420 s38 SNP 45% 385 s35 REF 60% 421 s38 REF 50% 386 s36 SNP 55% 422 s38 SNP 45% 387 s36 REF 50% 423 s39 SNP 30% 388 s36 SNP 45% 424 s39 SNP 30% 389 s36 REF 40% 425 s39 SNP 45% 390 s36 REF 50% 426 s39 REF 40% 391 s36 SNP 55% 427 s39 SNP 35% 392 s36 REF 40% 428 s39 REF 30% 393 s36 SNP 45% 429 s39 SNP 30% 394 s37 REF 65% 430 s40 SNP 40% 395 s37 SNP 60% 431 s40 REF 45% 396 s37 SNP 55% 432 s40 SNP 40% 397 s37 REF 60% 433 s40 REF 45% 398 s37 SNP 55% 434 s40 REF 40% 399 s37 REF 60% 435 s40 SNP 35% 400 s37 REF 55% 436 s40 SNP 40% 401 s37 SNP 50% 437 s40 REF 45% 402 s37 SNP 60% 438 s40 SNP 35% 439 s40 REF 40% 475 s46 REF 40% 440 s41 REF 70% 476 s46 SNP 40% 441 s41 SNP 70% 477 s46 REF 45% 442 s41 REF 60% 478 s46 SNP 45% 443 s41 REF 60% 479 s46 SNP 50% 444 s41 REF 70% 480 s46 REF 50% 445 s41 SNP 60% 481 s46 SNP 40% 446 s41 REF 60% 482 s46 REF 40% 447 s41 REF 60% 483 s46 SNP 50% 448 s41 SNP 60% 484 s46 SNP 35% 449 s41 REF 65% 485 s46 REF 35% 450 s41 SNP 65% 486 s47 SNP 65% 451 s41 SNP 70% 487 s47 SNP 65% 452 s41 SNP 65% 488 s47 SNP 65% 453 s41 REF 65% 489 s47 SNP 65% 454 s41 REF 65% 490 s47 SNP 70% 455 s41 REF 70% 491 s48 SNP 60% 456 s41 REF 70% 492 s48 SNP 60% 457 s41 REF 65% 493 s48 SNP 60% 458 s42 SNP 65% 494 s48 SNP 65% 459 s42 REF 70% 495 s48 SNP 65% 460 s42 SNP 55% 496 s49 SNP 55% 461 s42 REF 60% 497 s49 REF 60% 462 s42 REF 55% 498 s50 REF 70% 463 s42 SNP 50% 499 s50 SNP 65% 464 s43 SNP 45% 500 s51 SNP 55% 465 s43 REF 50% 501 s51 REF 60% 466 s43 SNP 55% 502 s51 REF 50% 467 s43 REF 60% 503 s51 SNP 45% 468 s43 SNP 50% 504 s51 REF 60% 469 s43 REF 55% 505 s51 SNP 55% 470 s43 SNP 70% 506 s52 REF 50% 471 s43 SNP 65% 507 s52 SNP 45% 472 s43 REF 70% 508 s53 REF 35% 473 s43 SNP 60% 509 s53 SNP 35% 474 s43 REF 65% 510 s53 REF 30% 511 s53 SNP 30% 547 s57 REF 30% 512 s53 REF 30% 548 s58 SNP 40% 513 s53 SNP 30% 549 s58 REF 45% 514 s54 SNP 45% 550 s58 REF 45% 515 s54 REF 40% 551 s58 SNP 40% 516 s54 REF 40% 552 s59 SNP 60% 517 s54 SNP 45% 553 s59 SNP 60% 518 s54 REF 40% 554 s59 SNP 60% 519 s54 SNP 45% 555 s59 SNP 55% 520 s54 REF 40% 556 s60 REF 65% 521 s54 SNP 45% 557 s60 SNP 60% 522 s54 SNP 45% 558 s60 REF 65% 523 s54 REF 40% 559 s60 SNP 60% 524 s55 SNP 45% 560 s60 SNP 50% 525 s55 REF 50% 561 s60 REF 55% 526 s55 SNP 50% 562 s60 SNP 50% 527 s55 REF 55% 563 s60 SNP 55% 528 s55 SNP 40% 564 s60 REF 60% 529 s55 REF 45% 565 s60 REF 55% 530 s55 SNP 40% 566 s61 REF 60% 531 s55 REF 45% 567 s61 SNP 60% 532 s56 SNP 35% 568 s61 REF 60% 533 s56 REF 40% 569 s61 REF 60% 534 s56 REF 40% 570 s61 REF 55% 535 s56 SNP 35% 571 s61 REF 75% 536 s56 SNP 40% 572 s61 REF 60% 537 s56 REF 45% 573 s61 REF 65% 538 s56 REF 55% 574 s61 SNP 70% 539 s56 SNP 50% 575 s61 SNP 55% 540 s56 REF 50% 576 s61 SNP 75% 541 s56 SNP 45% 577 s62 REF 55% 542 s56 REF 40% 578 s62 SNP 50% 543 s56 SNP 35% 579 s62 SNP 55% 544 s57 REF 30% 580 s62 SNP 45% 545 s57 REF 35% 581 s62 REF 50% 546 s57 SNP 30% 582 s62 REF 55% 583 s62 SNP 50% 619 s66 SNP 60% 584 s62 REF 55% 620 s66 REF 65% 585 s62 REF 55% 621 s66 SNP 60% 586 s63 REF 55% 622 s66 SNP 60% 587 s63 SNP 50% 623 s66 SNP 55% 588 s63 SNP 55% 624 s67 SNP 45% 589 s63 REF 60% 625 s67 REF 40% 590 s63 SNP 50% 626 s67 REF 30% 591 s63 REF 55% 627 s67 SNP 35% 592 s63 SNP 50% 628 s67 REF 45% 593 s63 REF 55% 629 s67 SNP 50% 594 s63 SNP 45% 630 s68 SNP 45% 595 s63 REF 50% 631 s68 REF 50% 596 s63 SNP 45% 632 s68 REF 55% 597 s63 REF 50% 633 s68 SNP 50% 598 s64 REF 45% 634 s69 SNP 70% 599 s64 SNP 40% 635 s69 REF 75% 600 s64 REF 55% 636 s69 REF 75% 601 s64 SNP 50% 637 s69 SNP 70% 602 s65 SNP 50% 638 s69 REF 75% 603 s65 REF 45% 639 s69 SNP 70% 604 s65 REF 45% 640 s69 SNP 70% 605 s65 SNP 50% 641 s69 REF 75% 606 s65 REF 45% 642 s69 SNP 70% 607 s65 SNP 50% 643 s69 SNP 75% 608 s66 SNP 75% 644 s69 REF 80% 609 s66 REF 80% 645 s69 REF 75% 610 s66 REF 60% 646 s69 SNP 70% 611 s66 SNP 55% 647 s69 REF 75% 612 s66 REF 60% 648 s69 REF 75% 613 s66 REF 65% 649 s69 SNP 70% 614 s66 SNP 60% 650 s69 SNP 65% 615 s66 SNP 60% 651 s69 REF 70% 616 s66 REF 65% 652 s70 REF 35% 617 s66 REF 65% 653 s70 SNP 30% 618 s66 REF 65% 654 s71 SNP 80% 655 s71 REF 80% 691 s74 SNP 40% 656 s71 REF 80% 692 s74 REF 45% 657 s71 SNP 80% 693 s74 REF 50% 658 s71 REF 80% 694 s74 SNP 45% 659 s71 SNP 80% 695 s74 REF 55% 660 s71 REF 85% 696 s74 SNP 40% 661 s71 SNP 85% 697 s74 REF 45% 662 s71 SNP 70% 698 s74 REF 45% 663 s71 SNP 75% 699 s74 SNP 40% 664 s71 REF 75% 700 s74 REF 45% 665 s71 REF 70% 701 s74 SNP 40% 666 s71 SNP 75% 702 s75 SNP 30% 667 s71 REF 75% 703 s75 SNP 40% 668 s72 REF 45% 704 s75 REF 35% 669 s72 SNP 40% 705 s76 REF 50% 670 s72 REF 45% 706 s76 SNP 45% 671 s72 REF 45% 707 s76 REF 45% 672 s72 REF 50% 708 s76 SNP 40% 673 s72 SNP 45% 709 s76 SNP 40% 674 s72 SNP 40% 710 s76 REF 45% 675 s72 REF 45% 711 s76 SNP 40% 676 s72 REF 50% 712 s76 REF 45% 677 s72 SNP 45% 713 s76 REF 50% 678 s73 REF 55% 714 s76 SNP 45% 679 s73 SNP 50% 715 s1 BOTH 40% 680 s73 SNP 50% 716 s1 BOTH 40% 681 s73 REF 55% 717 s2 BOTH 70% 682 s73 REF 60% 718 s3 BOTH 55% 683 s73 SNP 55% 719 s3 BOTH 60% 684 s73 SNP 50% 720 s3 BOTH 50% 685 s73 REF 55% 721 s3 BOTH 50% 686 s73 REF 55% 722 s4 BOTH 50% 687 s73 SNP 50% 723 s4 BOTH 50% 688 s74 SNP 50% 724 s4 BOTH 50% 689 s74 REF 45% 725 s4 BOTH 40% 690 s74 SNP 40% 726 s5 BOTH 60% 727 s5 BOTH 55% 763 s19 BOTH 55% 728 s6 BOTH 45% 764 s19 BOTH 60% 729 s7 BOTH 60% 765 s19 BOTH 40% 730 s7 BOTH 60% 766 s21, s22 BOTH, BOTH 55% 731 s8 BOTH 50% 767 s21, s22 REF, REF 60% 732 s8 BOTH 50% 768 s21, s22 REF, REF 65% 733 s8 BOTH 50% 769 s21, s22 REF, REF 65% 734 s9 BOTH 65% 770 s21, s22 REF, REF 65% 735 s9 BOTH 60% 771 s21, s22 REF, REF 70% 736 s10 BOTH 40% 772 s21, s22 REF, REF 60% 737 s10 BOTH 35% 773 s21, s22 REF, REF 70% 738 s11 BOTH 45% 774 s21, s22 REF, REF 65% 739 s12 BOTH 45% 775 s23 BOTH 55% 740 s12 BOTH 50% 776 s23 BOTH 55% 741 s12 BOTH 65% 777 s24 BOTH 60% 742 s13 BOTH 70% 778 s24 BOTH 65% 743 s13 BOTH 60% 779 s25 BOTH 60% 744 s13 BOTH 55% 780 s25 BOTH 60% 745 s14 BOTH 50% 781 s26 BOTH 60% 746 s14 BOTH 60% 782 s26 BOTH 35% 747 s14 BOTH 50% 783 s27 BOTH 50% 748 s14 BOTH 65% 784 s28 BOTH 60% 749 s14 BOTH 50% 785 s28 BOTH 55% 750 s15 BOTH 70% 786 s28 BOTH 55% 751 s15 BOTH 65% 787 s28 BOTH 45% 752 s15 BOTH 75% 788 s29 BOTH 50% 753 s16 BOTH 70% 789 s29 BOTH 45% 754 s16 BOTH 60% 790 s29 BOTH 40% 755 s17 BOTH 60% 791 s30 BOTH 60% 756 s17 BOTH 65% 792 s30 BOTH 60% 757 s17 BOTH 50% 793 s31 BOTH 40% 758 s18 BOTH 50% 794 s31 BOTH 35% 759 s18 BOTH 50% 795 s32 BOTH 70% 760 s18 BOTH 55% 796 s32 BOTH 65% 761 s18 BOTH 50% 797 s33 BOTH 50% 762 s19 BOTH 40% 798 s33 BOTH 60% 799 s34 BOTH 50% 835 s41 BOTH 65% 800 s34 BOTH 55% 836 s41 BOTH 50% 801 s34 BOTH 70% 837 s41 BOTH 50% 802 s34 BOTH 70% 838 s42 BOTH 75% 803 s35 BOTH 55% 839 s42 BOTH 75% 804 s35 BOTH 55% 840 s42 BOTH 40% 805 s35 BOTH 55% 841 s44 BOTH 35% 806 s35 BOTH 50% 842 s44 BOTH 35% 807 s35 BOTH 50% 843 s44 BOTH 40% 808 s35 BOTH 60% 844 s44 BOTH 30% 809 s35 BOTH 55% 845 s44 BOTH 40% 810 s35 BOTH 50% 846 s44 BOTH 35% 811 s35 BOTH 50% 847 s44 BOTH 35% 812 s35 BOTH 60% 848 s45 BOTH 50% 813 s35 BOTH 55% 849 s45 BOTH 55% 814 s35 BOTH 60% 850 s45 BOTH 50% 815 s35 BOTH 65% 851 s45 BOTH 50% 816 s35 BOTH 55% 852 s45 BOTH 50% 817 s35 BOTH 55% 853 s45 BOTH 55% 818 s36 BOTH 45% 854 s45 BOTH 50% 819 s36 BOTH 40% 855 s45 BOTH 55% 820 s36 BOTH 40% 856 s45 BOTH 55% 821 s37 BOTH 65% 857 s45 BOTH 55% 822 s37 BOTH 80% 858 s45 BOTH 55% 823 s37 BOTH 75% 859 s45 BOTH 50% 824 s37 BOTH 65% 860 s45 BOTH 50% 825 s38 BOTH 50% 861 s45 BOTH 50% 826 s38 BOTH 55% 862 s46, s27 REF, BOTH 50% 827 s38 BOTH 55% 863 s46, s27 REF, BOTH 45% 828 s39 BOTH 45% 864 s46, s27 BOTH, REF 55% 829 s39 BOTH 35% 865 s46, s27 REF, REF 50% 830 s40 BOTH 45% 866 s46 BOTH 35% 831 s40 BOTH 45% 867 s46 BOTH 35% 832 s40 BOTH 50% 868 s47 BOTH 55% 833 s41 BOTH 70% 869 s48, s47 REF, BOTH 65% 834 s41 BOTH 65% 870 s48, s47 REF, REF 65% 871 s48, s47 REF, REF 70% 907 s63 BOTH 60% 872 s48, s47 REF, REF 70% 908 s64 BOTH 60% 873 s48, s47 REF, REF 65% 909 s64 BOTH 55% 874 s48, s47 REF, REF 65% 910 s65 BOTH 50% 875 s48, s47 REF, REF 65% 911 s65 BOTH 40% 876 s48, s47 REF, REF 65% 912 s66 BOTH 65% 877 s48, s47 REF, REF 60% 913 s66 BOTH 60% 878 s48, s47 REF, REF 70% 914 s67 BOTH 30% 879 s48, s47 REF, REF 70% 915 s67 BOTH 45% 880 s48, s47 REF, REF 75% 916 s67 BOTH 45% 881 s48, s47 REF, REF 75% 917 s68 BOTH 45% 882 s48, s47 REF, REF 70% 918 s68 BOTH 50% 883 s48, s47 REF, REF 65% 919 s68 BOTH 55% 884 s49 BOTH 55% 920 s68 BOTH 55% 885 s50 BOTH 60% 921 s69 BOTH 65% 886 s50 BOTH 60% 922 s70 BOTH 35% 887 s52 BOTH 45% 923 s70 BOTH 45% 888 s52 BOTH 70% 924 s70 BOTH 45% 889 s53 BOTH 55% 925 s70 BOTH 45% 890 s53 BOTH 55% 926 s71 BOTH 60% 891 s54 BOTH 60% 927 s71 BOTH 65% 892 s55 BOTH 45% 928 s72 BOTH 40% 893 s55 BOTH 35% 929 s72 BOTH 50% 894 s56 BOTH 30% 930 s72 BOTH 45% 895 s56 BOTH 55% 931 s72 BOTH 40% 896 s56 BOTH 50% 932 s73 BOTH 60% 897 s57 BOTH 40% 933 s73 BOTH 60% 898 s57 BOTH 35% 934 s73 BOTH 65% 899 s58 BOTH 45% 935 s74 BOTH 45% 900 s58 BOTH 50% 936 s74 BOTH 45% 901 s59 BOTH 50% 937 s75 BOTH 50% 902 s60 BOTH 65% 938 s75 BOTH 55% 903 s60 BOTH 65% 939 s75 BOTH 30% 904 s61 BOTH 80% 940 s76 BOTH 35% 905 s61 BOTH 80% 941 s76 BOTH 50% 906 s62 BOTH 50% 942 s77 BOTH 65% 943 s77 BOTH 70% 979 s1 SNP 50% 944 s77 BOTH 30% 980 s1 REF 45% 945 s78 BOTH 40% 981 s1 REF 40% 946 s78 BOTH 30% 982 s1 SNP 45% 947 s78 BOTH 30% 983 s1 REF 45% 948 s79 REF 30% 984 s1 SNP 50% 949 s79 REF 40% 985 s1 SNP 55% 950 s79 REF 30% 986 s1 REF 50% 951 s79 REF 35% 987 s1 REF 50% 952 s80 SNP 45% 988 s1 SNP 55% 953 s80 SNP 35% 989 s1 REF 50% 954 s80 SNP 45% 990 s1 REF 35% 955 s80 SNP 45% 991 s1 SNP 40% 956 s80 SNP 45% 992 s81 SNP 50% 957 s80 SNP 45% 993 s2 REF 70% 958 s80 SNP 40% 994 s2 SNP 65% 959 s80 SNP 40% 995 s2 SNP 60% 960 s1 REF 45% 996 s2 REF 65% 961 s1 SNP 50% 997 s2 SNP 50% 962 s1 SNP 50% 998 s2 REF 55% 963 s1 REF 45% 999 s2 REF 65% 964 s1 REF 50% 1000 s2 SNP 60% 965 s1 SNP 55% 1001 s2 REF 60% 966 s1 SNP 40% 1002 s2 SNP 55% 967 s1 REF 35% 1003 s2 SNP 55% 968 s1 REF 45% 1004 s2 REF 60% 969 s1 SNP 50% 1005 s3 SNP 60% 970 s1 REF 40% 1006 s3 REF 60% 971 s1 SNP 45% 1007 s3 REF 55% 972 s1 REF 45% 1008 s3 SNP 50% 973 s1 REF 50% 1009 s3 REF 50% 974 s1 SNP 55% 1010 s3 SNP 65% 975 s1 SNP 50% 1011 s3 REF 65% 976 s1 SNP 55% 1012 s3 SNP 65% 977 s1 SNP 55% 1013 s3 REF 65% 978 s1 REF 50% 1014 s3 REF 65% 1015 s3 SNP 65% 1051 s4 SNP 50% 1016 s3 SNP 55% 1052 s4 REF 40% 1017 s3 REF 55% 1053 s4 REF 50% 1018 s3 REF 55% 1054 s4 REF 45% 1019 s3 SNP 55% 1055 s4 SNP 50% 1020 s3 REF 55% 1056 s4 SNP 50% 1021 s3 REF 60% 1057 s4 REF 45% 1022 s3 SNP 60% 1058 s4 SNP 45% 1023 s3 SNP 60% 1059 s4 SNP 45% 1024 s3 SNP 55% 1060 s4 REF 40% 1025 s3 REF 55% 1061 s4 REF 35% 1026 s3 SNP 55% 1062 s4 SNP 40% 1027 s3 REF 55% 1063 s4 REF 40% 1028 s3 SNP 55% 1064 s4 SNP 45% 1029 s3 REF 65% 1065 s4 SNP 45% 1030 s3 SNP 65% 1066 s4 REF 40% 1031 s3 REF 60% 1067 s4 SNP 45% 1032 s3 SNP 60% 1068 s4 REF 40% 1033 s3 REF 60% 1069 s5 SNP 60% 1034 s3 SNP 60% 1070 s5 REF 60% 1035 s3 REF 60% 1071 s5 SNP 65% 1036 s3 SNP 55% 1072 s5 REF 65% 1037 s4 SNP 50% 1073 s5 REF 65% 1038 s4 REF 45% 1074 s5 REF 70% 1039 s4 REF 50% 1075 s5 SNP 70% 1040 s4 SNP 55% 1076 s5 SNP 65% 1041 s4 REF 40% 1077 s5 SNP 65% 1042 s4 SNP 45% 1078 s5 SNP 65% 1043 s4 SNP 55% 1079 s5 REF 65% 1044 s4 SNP 50% 1080 s5 REF 65% 1045 s4 REF 45% 1081 s5 SNP 65% 1046 s4 SNP 45% 1082 s5 SNP 65% 1047 s4 REF 40% 1083 s5 REF 65% 1048 s4 REF 45% 1084 s5 SNP 65% 1049 s4 SNP 50% 1085 s5 REF 65% 1050 s4 REF 45% 1086 s5 SNP 65% 1087 s5 REF 65% 1123 s6 REF 50% 1088 s5 REF 65% 1124 s6 SNP 55% 1089 s5 REF 60% 1125 s6 REF 40% 1090 s5 SNP 60% 1126 s6 SNP 45% 1091 s5 SNP 60% 1127 s6 REF 40% 1092 s5 REF 60% 1128 s6 SNP 45% 1093 s5 REF 60% 1129 s6 SNP 45% 1094 s5 SNP 60% 1130 s6 REF 40% 1095 s5 REF 65% 1131 s6 REF 40% 1096 s5 SNP 65% 1132 s6 SNP 45% 1097 s5 REF 65% 1133 s7 SNP 55% 1098 s5 SNP 65% 1134 s7 SNP 40% 1099 s6 REF 40% 1135 s7 REF 45% 1100 s6 SNP 45% 1136 s7 REF 55% 1101 s6 SNP 50% 1137 s7 SNP 50% 1102 s6 REF 45% 1138 s7 REF 55% 1103 s6 REF 40% 1139 s7 SNP 50% 1104 s6 SNP 45% 1140 s7 SNP 45% 1105 s6 SNP 45% 1141 s7 REF 50% 1106 s6 REF 40% 1142 s7 SNP 50% 1107 s6 REF 50% 1143 s7 REF 55% 1108 s6 SNP 55% 1144 s7 REF 50% 1109 s6 SNP 55% 1145 s7 SNP 45% 1110 s6 REF 50% 1146 s7 SNP 60% 1111 s6 REF 45% 1147 s7 REF 65% 1112 s6 SNP 50% 1148 s7 SNP 55% 1113 s6 REF 40% 1149 s7 SNP 50% 1114 s6 SNP 45% 1150 s7 REF 55% 1115 s6 SNP 55% 1151 s8 REF 45% 1116 s6 REF 50% 1152 s8 REF 35% 1117 s6 REF 50% 1153 s8 SNP 40% 1118 s6 SNP 55% 1154 s8 REF 40% 1119 s6 SNP 45% 1155 s8 SNP 45% 1120 s6 REF 40% 1156 s8 REF 40% 1121 s6 SNP 55% 1157 s8 SNP 45% 1122 s6 REF 50% 1158 s8 SNP 50% 1159 s8 REF 45% 1195 s9 REF 70% 1160 s8 SNP 50% 1196 s9 SNP 65% 1161 s8 SNP 50% 1197 s9 REF 65% 1162 s8 REF 45% 1198 s9 SNP 60% 1163 s8 SNP 45% 1199 s9 REF 60% 1164 s8 REF 40% 1200 s9 SNP 55% 1165 s8 REF 35% 1201 s9 REF 55% 1166 s8 SNP 40% 1202 s9 SNP 60% 1167 s8 SNP 50% 1203 s9 REF 65% 1168 s8 REF 45% 1204 s9 REF 70% 1169 s8 SNP 45% 1205 s9 SNP 65% 1170 s8 REF 40% 1206 s9 SNP 65% 1171 s8 SNP 45% 1207 s9 REF 70% 1172 s8 REF 40% 1208 s9 REF 65% 1173 s8 REF 40% 1209 s9 SNP 60% 1174 s8 SNP 45% 1210 s9 REF 65% 1175 s8 SNP 40% 1211 s9 SNP 60% 1176 s8 REF 35% 1212 s9 REF 65% 1177 s8 SNP 40% 1213 s9 SNP 60% 1178 s8 REF 35% 1214 s9 SNP 50% 1179 s8 SNP 50% 1215 s9 SNP 55% 1180 s8 REF 45% 1216 s9 REF 60% 1181 s8 SNP 50% 1217 s10 REF 30% 1182 s8 REF 45% 1218 s10 SNP 30% 1183 s8 REF 40% 1219 s10 REF 35% 1184 s8 SNP 45% 1220 s10 REF 40% 1185 s9 SNP 60% 1221 s10 SNP 35% 1186 s9 REF 65% 1222 s10 REF 30% 1187 s9 REF 60% 1223 s10 REF 30% 1188 s9 SNP 55% 1224 s10 SNP 30% 1189 s9 REF 55% 1225 s10 REF 35% 1190 s9 SNP 50% 1226 s10 REF 35% 1191 s9 SNP 60% 1227 s10 SNP 30% 1192 s9 REF 65% 1228 s10 REF 35% 1193 s9 SNP 50% 1229 s10 REF 30% 1194 s9 REF 55% 1230 s10 SNP 30% 1231 s11 SNP 45% 1267 s12 REF 65% 1232 s11 SNP 45% 1268 s12 REF 65% 1233 s11 REF 50% 1269 s12 SNP 70% 1234 s11 SNP 50% 1270 s12 REF 65% 1235 s11 REF 55% 1271 s12 SNP 70% 1236 s11 SNP 45% 1272 s12 SNP 70% 1237 s11 REF 50% 1273 s12 REF 65% 1238 s11 SNP 40% 1274 s12 REF 55% 1239 s11 REF 45% 1275 s12 SNP 60% 1240 s11 REF 50% 1276 s12 SNP 60% 1241 s11 SNP 45% 1277 s12 SNP 70% 1242 s11 REF 35% 1278 s12 REF 65% 1243 s11 SNP 30% 1279 s12 SNP 75% 1244 s11 REF 50% 1280 s12 REF 70% 1245 s11 SNP 45% 1281 s12 REF 55% 1246 s11 REF 45% 1282 s12 REF 60% 1247 s11 SNP 40% 1283 s12 SNP 65% 1248 s11 REF 55% 1284 s13 SNP 70% 1249 s11 SNP 50% 1285 s13 SNP 65% 1250 s11 REF 50% 1286 s13 REF 70% 1251 s11 SNP 45% 1287 s13 SNP 70% 1252 s11 REF 40% 1288 s13 REF 75% 1253 s11 SNP 35% 1289 s13 SNP 65% 1254 s11 SNP 35% 1290 s13 REF 70% 1255 s11 REF 40% 1291 s13 REF 70% 1256 s11 SNP 30% 1292 s13 SNP 65% 1257 s11 REF 35% 1293 s13 SNP 65% 1258 s12 REF 60% 1294 s13 REF 70% 1259 s12 SNP 65% 1295 s13 REF 70% 1260 s12 SNP 70% 1296 s13 SNP 65% 1261 s12 REF 65% 1297 s13 SNP 70% 1262 s12 REF 65% 1298 s13 REF 75% 1263 s12 SNP 70% 1299 s13 SNP 70% 1264 s12 SNP 65% 1300 s13 REF 75% 1265 s12 REF 60% 1301 s13 SNP 60% 1266 s12 SNP 70% 1302 s13 REF 65% 1303 s13 REF 75% 1339 s14 SNP 65% 1304 s13 REF 65% 1340 s14 SNP 65% 1305 s13 REF 75% 1341 s14 REF 60% 1306 s13 SNP 70% 1342 s14 REF 60% 1307 s13 REF 75% 1343 s14 SNP 65% 1308 s13 SNP 70% 1344 s14 REF 60% 1309 s13 REF 75% 1345 s14 SNP 65% 1310 s13 SNP 70% 1346 s14 REF 60% 1311 s13 SNP 60% 1347 s14 REF 60% 1312 s13 REF 70% 1348 s14 REF 50% 1313 s13 SNP 65% 1349 s14 REF 65% 1314 s13 SNP 65% 1350 s14 REF 60% 1315 s13 REF 70% 1351 s14 SNP 65% 1316 s13 REF 75% 1352 s14 REF 65% 1317 s13 SNP 70% 1353 s14 REF 65% 1318 s14 SNP 60% 1354 s14 REF 65% 1319 s14 REF 55% 1355 s14 SNP 70% 1320 s14 SNP 55% 1356 s14 REF 55% 1321 s14 REF 50% 1357 s14 REF 65% 1322 s14 REF 55% 1358 s14 REF 60% 1323 s14 REF 55% 1359 s14 REF 60% 1324 s14 REF 50% 1360 s14 REF 60% 1325 s14 SNP 65% 1361 s82 REF 30% 1326 s14 REF 60% 1362 s82 REF 40% 1327 s14 REF 65% 1363 s82 REF 65% 1328 s14 SNP 70% 1364 s82 REF 45% 1329 s14 SNP 70% 1365 s82 REF 60% 1330 s14 REF 65% 1366 s82 REF 60% 1331 s14 REF 55% 1367 s82 REF 30% 1332 s14 SNP 60% 1368 s82 SNP 30% 1333 s14 REF 65% 1369 s82 REF 55% 1334 s14 REF 55% 1370 s82 REF 40% 1335 s14 REF 65% 1371 s15 REF 65% 1336 s14 SNP 70% 1372 s15 SNP 65% 1337 s14 REF 60% 1373 s15 REF 80% 1338 s14 REF 60% 1374 s15 SNP 80% 1375 s15 REF 70% 1411 s16 REF 50% 1376 s15 SNP 70% 1412 s16 SNP 60% 1377 s15 REF 70% 1413 s16 REF 65% 1378 s15 SNP 70% 1414 s16 REF 65% 1379 s15 REF 75% 1415 s16 SNP 60% 1380 s15 SNP 75% 1416 s16 SNP 60% 1381 s15 REF 85% 1417 s16 REF 65% 1382 s15 SNP 85% 1418 s16 REF 60% 1383 s15 REF 80% 1419 s16 SNP 55% 1384 s15 SNP 80% 1420 s16 SNP 55% 1385 s15 REF 80% 1421 s16 REF 60% 1386 s15 REF 75% 1422 s16 REF 65% 1387 s15 SNP 75% 1423 s16 SNP 60% 1388 s15 REF 70% 1424 s16 REF 50% 1389 s15 SNP 70% 1425 s16 SNP 45% 1390 s15 SNP 80% 1426 s16 SNP 60% 1391 s15 SNP 75% 1427 s16 REF 55% 1392 s15 REF 75% 1428 s16 SNP 50% 1393 s15 SNP 80% 1429 s17 SNP 60% 1394 s15 REF 80% 1430 s17 REF 65% 1395 s15 SNP 70% 1431 s17 SNP 60% 1396 s15 REF 70% 1432 s17 SNP 60% 1397 s16 SNP 55% 1433 s17 REF 65% 1398 s16 REF 60% 1434 s17 REF 65% 1399 s16 REF 60% 1435 s17 SNP 50% 1400 s16 SNP 55% 1436 s17 REF 55% 1401 s16 SNP 40% 1437 s17 REF 65% 1402 s16 REF 45% 1438 s17 SNP 60% 1403 s16 SNP 45% 1439 s17 SNP 60% 1404 s16 SNP 55% 1440 s17 REF 65% 1405 s16 REF 60% 1441 s17 SNP 60% 1406 s16 SNP 50% 1442 s17 REF 65% 1407 s16 REF 55% 1443 s17 SNP 45% 1408 s16 SNP 60% 1444 s17 REF 50% 1409 s16 REF 65% 1445 s17 REF 70% 1410 s16 REF 65% 1446 s17 SNP 65% 1447 s17 REF 65% 1483 s18 REF 55% 1448 s17 SNP 60% 1484 s18 SNP 50% 1449 s17 REF 50% 1485 s19 REF 50% 1450 s17 SNP 60% 1486 s19 SNP 45% 1451 s17 REF 65% 1487 s19 REF 50% 1452 s17 REF 65% 1488 s19 SNP 45% 1453 s17 SNP 60% 1489 s19 REF 60% 1454 s17 SNP 45% 1490 s19 SNP 55% 1455 s17 SNP 60% 1491 s19 REF 55% 1456 s17 REF 65% 1492 s19 SNP 50% 1457 s18 REF 55% 1493 s19 REF 45% 1458 s18 SNP 50% 1494 s19 REF 50% 1459 s18 REF 55% 1495 s19 SNP 45% 1460 s18 SNP 50% 1496 s19 SNP 40% 1461 s18 REF 55% 1497 s19 REF 45% 1462 s18 SNP 50% 1498 s19 SNP 55% 1463 s18 REF 50% 1499 s19 SNP 45% 1464 s18 SNP 45% 1500 s19 REF 50% 1465 s18 SNP 45% 1501 s19 REF 60% 1466 s18 REF 50% 1502 s19 SNP 55% 1467 s18 SNP 45% 1503 s19 REF 60% 1468 s18 SNP 50% 1504 s19 SNP 45% 1469 s18 REF 55% 1505 s19 REF 50% 1470 s18 SNP 45% 1506 s19 SNP 40% 1471 s18 REF 55% 1507 s19 REF 45% 1472 s18 SNP 50% 1508 s19 SNP 50% 1473 s18 REF 55% 1509 s19 REF 55% 1474 s18 SNP 50% 1510 s19 SNP 40% 1475 s18 REF 50% 1511 s19 SNP 45% 1476 s18 SNP 45% 1512 s19 REF 50% 1477 s18 REF 50% 1513 s20 REF 45% 1478 s18 REF 55% 1514 s22 SNP 65% 1479 s18 SNP 50% 1515 s22 SNP 65% 1480 s18 REF 55% 1516 s22 SNP 65% 1481 s18 SNP 50% 1517 s22 SNP 70% 1482 s18 REF 50% 1518 s22 SNP 60% 1519 s21 SNP 65% 1555 s23 REF 55% 1520 s21 SNP 70% 1556 s23 REF 50% 1521 s21 SNP 70% 1557 s23 REF 50% 1522 s21 SNP 70% 1558 s23 SNP 55% 1523 s21 SNP 75% 1559 s24 SNP 60% 1524 s21 SNP 75% 1560 s24 REF 65% 1525 s21 SNP 65% 1561 s24 REF 60% 1526 s21 SNP 70% 1562 s24 SNP 55% 1527 s23 REF 50% 1563 s24 SNP 50% 1528 s23 SNP 55% 1564 s24 REF 55% 1529 s23 SNP 50% 1565 s24 SNP 50% 1530 s23 REF 45% 1566 s24 REF 55% 1531 s23 REF 45% 1567 s24 SNP 50% 1532 s23 SNP 50% 1568 s24 REF 55% 1533 s23 SNP 60% 1569 s24 SNP 55% 1534 s23 REF 55% 1570 s24 SNP 45% 1535 s23 REF 50% 1571 s24 REF 50% 1536 s23 SNP 55% 1572 s24 REF 60% 1537 s23 REF 55% 1573 s24 SNP 50% 1538 s23 SNP 60% 1574 s24 REF 55% 1539 s23 SNP 55% 1575 s24 REF 55% 1540 s23 SNP 65% 1576 s24 SNP 50% 1541 s23 REF 60% 1577 s24 REF 50% 1542 s23 REF 50% 1578 s24 SNP 45% 1543 s23 SNP 55% 1579 s24 REF 65% 1544 s23 SNP 60% 1580 s24 SNP 60% 1545 s23 REF 55% 1581 s24 REF 55% 1546 s23 SNP 55% 1582 s24 SNP 50% 1547 s23 REF 50% 1583 s24 REF 55% 1548 s23 REF 60% 1584 s24 SNP 45% 1549 s23 SNP 65% 1585 s24 REF 50% 1550 s23 SNP 55% 1586 s24 SNP 50% 1551 s23 REF 50% 1587 s24 SNP 50% 1552 s23 REF 55% 1588 s24 REF 55% 1553 s23 SNP 60% 1589 s25 SNP 55% 1554 s23 SNP 60% 1590 s25 REF 60% 1591 s25 SNP 45% 1627 s26 REF 65% 1592 s25 REF 50% 1628 s27 REF 50% 1593 s25 REF 60% 1629 s27 SNP 45% 1594 s25 SNP 55% 1630 s27 SNP 45% 1595 s25 SNP 50% 1631 s27 REF 50% 1596 s25 REF 55% 1632 s27 SNP 50% 1597 s25 REF 65% 1633 s27 SNP 45% 1598 s25 SNP 60% 1634 s27 SNP 50% 1599 s25 SNP 55% 1635 s27 REF 55% 1600 s25 REF 60% 1636 s27 SNP 50% 1601 s25 REF 60% 1637 s27 SNP 50% 1602 s25 SNP 55% 1638 s27 SNP 45% 1603 s25 REF 60% 1639 s27 REF 50% 1604 s25 SNP 55% 1640 s27 SNP 45% 1605 s26 SNP 45% 1641 s27 REF 50% 1606 s26 REF 50% 1642 s27 REF 55% 1607 s26 REF 55% 1643 s27 SNP 50% 1608 s26 SNP 50% 1644 s27 REF 55% 1609 s26 REF 50% 1645 s27 SNP 50% 1610 s26 SNP 45% 1646 s27 REF 55% 1611 s26 SNP 45% 1647 s27 SNP 45% 1612 s26 REF 50% 1648 s27 REF 50% 1613 s26 REF 40% 1649 s27 SNP 45% 1614 s26 SNP 35% 1650 s27 REF 55% 1615 s26 SNP 60% 1651 s27 SNP 50% 1616 s26 REF 60% 1652 s27 SNP 45% 1617 s26 SNP 55% 1653 s27 REF 55% 1618 s26 REF 65% 1654 s27 SNP 50% 1619 s26 SNP 60% 1655 s27 SNP 50% 1620 s26 REF 50% 1656 s27 REF 55% 1621 s26 SNP 45% 1657 s27 REF 55% 1622 s26 REF 65% 1658 s27 SNP 50% 1623 s26 SNP 60% 1659 s28 REF 60% 1624 s26 SNP 65% 1660 s28 SNP 65% 1625 s26 REF 70% 1661 s28 SNP 65% 1626 s26 SNP 60% 1662 s28 REF 60% 1663 s28 SNP 65% 1699 s29 REF 55% 1664 s28 REF 60% 1700 s29 SNP 45% 1665 s28 REF 65% 1701 s29 REF 50% 1666 s28 SNP 70% 1702 s29 SNP 50% 1667 s28 REF 60% 1703 s29 REF 55% 1668 s28 SNP 65% 1704 s29 REF 45% 1669 s28 REF 55% 1705 s29 SNP 40% 1670 s28 SNP 60% 1706 s29 REF 55% 1671 s28 SNP 70% 1707 s29 SNP 50% 1672 s28 REF 65% 1708 s29 SNP 45% 1673 s28 REF 55% 1709 s29 SNP 50% 1674 s28 SNP 60% 1710 s29 REF 55% 1675 s28 SNP 55% 1711 s29 REF 50% 1676 s28 REF 50% 1712 s29 SNP 45% 1677 s28 SNP 65% 1713 s29 SNP 45% 1678 s28 REF 60% 1714 s29 REF 50% 1679 s28 SNP 60% 1715 s29 REF 50% 1680 s28 REF 65% 1716 s29 REF 50% 1681 s28 SNP 70% 1717 s29 SNP 45% 1682 s28 SNP 70% 1718 s29 REF 55% 1683 s28 REF 65% 1719 s29 SNP 50% 1684 s28 REF 60% 1720 s29 REF 55% 1685 s28 SNP 65% 1721 s29 SNP 50% 1686 s28 REF 55% 1722 s29 SNP 40% 1687 s28 SNP 55% 1723 s29 REF 45% 1688 s28 SNP 55% 1724 s29 REF 50% 1689 s28 SNP 60% 1725 s29 SNP 45% 1690 s28 REF 55% 1726 s29 REF 50% 1691 s83 SNP 45% 1727 s29 SNP 45% 1692 s83 SNP 50% 1728 s30 REF 60% 1693 s83 SNP 40% 1729 s30 REF 60% 1694 s83 SNP 45% 1730 s30 SNP 65% 1695 s83 SNP 45% 1731 s30 SNP 65% 1696 s83 SNP 50% 1732 s30 SNP 70% 1697 s83 SNP 50% 1733 s30 REF 65% 1698 s29 SNP 50% 1734 s30 REF 70% 1735 s30 SNP 75% 1771 s31 REF 40% 1736 s30 SNP 65% 1772 s31 SNP 40% 1737 s30 SNP 60% 1773 s31 REF 40% 1738 s30 SNP 65% 1774 s31 SNP 40% 1739 s30 SNP 65% 1775 s31 SNP 60% 1740 s30 SNP 70% 1776 s31 REF 60% 1741 s30 REF 65% 1777 s31 REF 60% 1742 s30 SNP 65% 1778 s31 REF 50% 1743 s30 SNP 60% 1779 s31 SNP 50% 1744 s30 SNP 65% 1780 s31 SNP 60% 1745 s30 REF 60% 1781 s84 SNP 65% 1746 s30 SNP 65% 1782 s84 REF 65% 1747 s31 REF 35% 1783 s84 REF 75% 1748 s31 SNP 35% 1784 s84 SNP 65% 1749 s31 REF 50% 1785 s84 REF 70% 1750 s31 SNP 50% 1786 s84 SNP 60% 1751 s31 SNP 40% 1787 s84 REF 70% 1752 s31 REF 40% 1788 s84 SNP 70% 1753 s31 REF 70% 1789 s84 SNP 60% 1754 s31 REF 55% 1790 s32 SNP 50% 1755 s31 SNP 55% 1791 s32 REF 45% 1756 s31 SNP 40% 1792 s32 REF 50% 1757 s31 REF 40% 1793 s32 SNP 50% 1758 s31 REF 40% 1794 s32 REF 45% 1759 s31 SNP 40% 1795 s32 REF 55% 1760 s31 SNP 70% 1796 s32 SNP 60% 1761 s31 SNP 50% 1797 s32 SNP 70% 1762 s31 REF 50% 1798 s32 SNP 55% 1763 s31 SNP 55% 1799 s32 REF 50% 1764 s31 REF 55% 1800 s32 SNP 55% 1765 s31 SNP 50% 1801 s32 REF 50% 1766 s31 REF 50% 1802 s32 REF 65% 1767 s31 SNP 70% 1803 s32 SNP 70% 1768 s31 REF 70% 1804 s32 SNP 55% 1769 s31 REF 60% 1805 s32 REF 50% 1770 s31 SNP 60% 1806 s32 REF 65% 1807 s32 REF 65% 1843 s34 REF 70% 1808 s32 SNP 70% 1844 s34 REF 60% 1809 s32 REF 55% 1845 s34 SNP 55% 1810 s32 SNP 60% 1846 s34 REF 65% 1811 s32 REF 50% 1847 s34 SNP 60% 1812 s32 SNP 55% 1848 s34 SNP 60% 1813 s32 SNP 55% 1849 s34 REF 65% 1814 s32 REF 50% 1850 s34 REF 65% 1815 s32 SNP 55% 1851 s34 SNP 60% 1816 s32 REF 50% 1852 s34 REF 50% 1817 s32 SNP 55% 1853 s34 REF 65% 1818 s32 SNP 55% 1854 s34 SNP 60% 1819 s32 REF 50% 1855 s34 REF 65% 1820 s32 SNP 60% 1856 s34 SNP 60% 1821 s32 REF 55% 1857 s34 REF 70% 1822 s33 REF 65% 1858 s34 SNP 65% 1823 s33 SNP 60% 1859 s34 REF 60% 1824 s33 REF 65% 1860 s34 SNP 55% 1825 s33 SNP 60% 1861 s34 REF 65% 1826 s33 REF 65% 1862 s34 SNP 60% 1827 s33 SNP 60% 1863 s34 SNP 55% 1828 s33 REF 70% 1864 s34 REF 60% 1829 s33 SNP 65% 1865 s34 SNP 60% 1830 s33 REF 70% 1866 s34 REF 65% 1831 s33 SNP 65% 1867 s34 SNP 60% 1832 s33 REF 70% 1868 s34 REF 65% 1833 s33 SNP 65% 1869 s34 SNP 55% 1834 s33 REF 60% 1870 s34 REF 60% 1835 s33 SNP 65% 1871 s34 SNP 45% 1836 s33 REF 70% 1872 s34 SNP 45% 1837 s33 SNP 60% 1873 s34 REF 50% 1838 s33 REF 65% 1874 s35 REF 70% 1839 s33 SNP 55% 1875 s35 REF 65% 1840 s34 SNP 65% 1876 s36 SNP 40% 1841 s34 SNP 60% 1877 s36 REF 35% 1842 s34 REF 65% 1878 s36 REF 40% 1879 s36 SNP 45% 1915 s37 SNP 65% 1880 s36 REF 35% 1916 s37 REF 70% 1881 s36 SNP 40% 1917 s37 SNP 65% 1882 s36 SNP 40% 1918 s37 SNP 65% 1883 s36 REF 50% 1919 s37 REF 70% 1884 s36 SNP 55% 1920 s37 SNP 65% 1885 s36 REF 35% 1921 s37 REF 70% 1886 s36 SNP 40% 1922 s37 SNP 70% 1887 s36 REF 35% 1923 s37 REF 75% 1888 s36 SNP 40% 1924 s37 SNP 60% 1889 s36 REF 40% 1925 s37 SNP 60% 1890 s36 SNP 45% 1926 s37 REF 65% 1891 s36 SNP 55% 1927 s37 REF 65% 1892 s36 REF 50% 1928 s37 REF 65% 1893 s36 SNP 40% 1929 s37 SNP 60% 1894 s36 REF 35% 1930 s85 SNP 45% 1895 s36 REF 35% 1931 s38 SNP 45% 1896 s36 REF 40% 1932 s38 REF 50% 1897 s36 SNP 45% 1933 s38 SNP 50% 1898 s36 SNP 45% 1934 s38 REF 55% 1899 s36 REF 40% 1935 s38 REF 50% 1900 s36 SNP 45% 1936 s38 REF 55% 1901 s36 REF 40% 1937 s38 SNP 50% 1902 s36 REF 50% 1938 s38 SNP 45% 1903 s36 SNP 55% 1939 s38 REF 50% 1904 s36 SNP 55% 1940 s38 SNP 45% 1905 s36 REF 50% 1941 s38 REF 50% 1906 s36 SNP 45% 1942 s38 SNP 45% 1907 s36 REF 40% 1943 s38 REF 55% 1908 s37 SNP 50% 1944 s38 SNP 50% 1909 s37 REF 55% 1945 s38 SNP 50% 1910 s37 SNP 55% 1946 s38 REF 55% 1911 s37 REF 60% 1947 s38 SNP 50% 1912 s37 REF 60% 1948 s38 REF 55% 1913 s37 SNP 55% 1949 s38 REF 55% 1914 s37 REF 70% 1950 s38 SNP 50% 1951 s38 REF 50% 1987 s40 REF 45% 1952 s38 SNP 45% 1988 s40 SNP 40% 1953 s38 SNP 50% 1989 s40 REF 45% 1954 s38 REF 55% 1990 s40 SNP 40% 1955 s38 SNP 50% 1991 s40 REF 40% 1956 s38 REF 55% 1992 s40 SNP 35% 1957 s38 REF 55% 1993 s40 SNP 35% 1958 s38 SNP 50% 1994 s40 REF 40% 1959 s38 REF 55% 1995 s40 REF 50% 1960 s38 SNP 50% 1996 s40 SNP 45% 1961 s39 REF 40% 1997 s40 REF 50% 1962 s39 SNP 45% 1998 s40 SNP 45% 1963 s39 REF 40% 1999 s40 SNP 30% 1964 s39 SNP 45% 2000 s40 REF 35% 1965 s39 REF 40% 2001 s40 REF 50% 1966 s39 SNP 45% 2002 s40 REF 35% 1967 s39 SNP 30% 2003 s40 SNP 30% 1968 s39 SNP 30% 2004 s40 REF 45% 1969 s39 SNP 30% 2005 s40 REF 40% 1970 s39 SNP 45% 2006 s40 SNP 35% 1971 s39 REF 40% 2007 s40 SNP 40% 1972 s39 REF 40% 2008 s40 REF 45% 1973 s39 SNP 45% 2009 s40 REF 45% 1974 s39 REF 30% 2010 s40 SNP 40% 1975 s39 SNP 35% 2011 s40 SNP 40% 1976 s39 SNP 30% 2012 s41 REF 65% 1977 s39 REF 30% 2013 s41 SNP 65% 1978 s39 SNP 35% 2014 s41 SNP 65% 1979 s39 SNP 35% 2015 s41 REF 65% 1980 s39 REF 30% 2016 s41 SNP 70% 1981 s39 SNP 30% 2017 s41 REF 65% 1982 s40 SNP 45% 2018 s41 REF 70% 1983 s40 SNP 35% 2019 s41 REF 70% 1984 s40 REF 40% 2020 s41 REF 65% 1985 s40 SNP 45% 2021 s41 REF 65% 1986 s40 REF 50% 2022 s41 REF 70% 2023 s41 SNP 70% 2059 s42 SNP 50% 2024 s41 REF 60% 2060 s42 REF 70% 2025 s41 REF 65% 2061 s42 SNP 65% 2026 s41 SNP 65% 2062 s42 REF 65% 2027 s41 REF 65% 2063 s42 SNP 60% 2028 s41 SNP 60% 2064 s42 REF 45% 2029 s41 REF 65% 2065 s42 REF 50% 2030 s41 SNP 65% 2066 s42 SNP 45% 2031 s41 REF 60% 2067 s42 SNP 65% 2032 s41 REF 65% 2068 s42 REF 70% 2033 s41 REF 60% 2069 s42 SNP 50% 2034 s41 REF 65% 2070 s42 REF 55% 2035 s41 REF 65% 2071 s42 SNP 50% 2036 s41 REF 65% 2072 s42 REF 55% 2037 s41 SNP 65% 2073 s42 SNP 45% 2038 s41 SNP 65% 2074 s42 REF 50% 2039 s41 REF 65% 2075 s42 REF 60% 2040 s41 REF 65% 2076 s42 SNP 55% 2041 s41 SNP 65% 2077 s42 REF 50% 2042 s41 REF 70% 2078 s42 REF 45% 2043 s41 REF 60% 2079 s42 SNP 40% 2044 s41 REF 65% 2080 s42 SNP 45% 2045 s41 SNP 65% 2081 s42 SNP 60% 2046 s41 REF 65% 2082 s42 REF 65% 2047 s41 SNP 60% 2083 s42 SNP 40% 2048 s41 REF 60% 2084 s42 REF 45% 2049 s41 SNP 60% 2085 s42 SNP 40% 2050 s41 REF 60% 2086 s42 REF 45% 2051 s41 REF 60% 2087 s42 SNP 40% 2052 s41 SNP 60% 2088 s43 SNP 65% 2053 s41 REF 60% 2089 s43 REF 70% 2054 s42 REF 70% 2090 s43 REF 50% 2055 s42 SNP 65% 2091 s43 SNP 45% 2056 s42 SNP 45% 2092 s43 REF 60% 2057 s42 REF 50% 2093 s43 SNP 55% 2058 s42 REF 55% 2094 s43 REF 55% 2095 s43 REF 70% 2131 s46 SNP 50% 2096 s43 SNP 65% 2132 s46 REF 45% 2097 s43 REF 75% 2133 s46 SNP 45% 2098 s43 SNP 70% 2134 s46 SNP 40% 2099 s43 SNP 70% 2135 s46 REF 40% 2100 s43 REF 70% 2136 s47 SNP 65% 2101 s43 SNP 65% 2137 s47 SNP 70% 2102 s43 SNP 50% 2138 s47 SNP 60% 2103 s43 REF 75% 2139 s47 SNP 70% 2104 s43 SNP 70% 2140 s47 SNP 65% 2105 s43 SNP 70% 2141 s47 SNP 65% 2106 s43 REF 75% 2142 s47 SNP 65% 2107 s43 REF 75% 2143 s47 SNP 65% 2108 s43 SNP 70% 2144 s47 SNP 60% 2109 s43 REF 65% 2145 s47 SNP 70% 2110 s43 SNP 60% 2146 s47 SNP 70% 2111 s46 SNP 45% 2147 s47 SNP 75% 2112 s46 REF 45% 2148 s47 SNP 75% 2113 s46 REF 40% 2149 s47 SNP 70% 2114 s46 SNP 40% 2150 s47 REF 60% 2115 s46 REF 50% 2151 s47 SNP 65% 2116 s46 SNP 50% 2152 s48 SNP 60% 2117 s46 REF 35% 2153 s48 SNP 60% 2118 s46 SNP 35% 2154 s48 SNP 60% 2119 s46 REF 45% 2155 s48 SNP 65% 2120 s46 SN 45% 2156 s48 SNP 70% 2121 s46 SNP 50% 2157 s48 REF 65% 2122 s46 SNP 45% 2158 s48 SNP 65% 2123 s46 SNP 50% 2159 s48 SNP 65% 2124 s46 REF 50% 2160 s48 SNP 65% 2125 s46 SNP 50% 2161 s48 SNP 65% 2126 s46 SNP 45% 2162 s48 SNP 65% 2127 s46 REF 45% 2163 s48 SNP 70% 2128 s46 SNP 50% 2164 s48 SNP 65% 2129 s46 REF 50% 2165 s48 SNP 70% 2130 s46 REF 50% 2166 s48 SNP 70% 2167 s48 SNP 60% 2203 s52 REF 55% 2168 s48 SNP 65% 2204 s52 SNP 55% 2169 s48 SNP 60% 2205 s52 REF 60% 2170 s49 SNP 45% 2206 s52 SNP 65% 2171 s49 SNP 55% 2207 s52 REF 70% 2172 s49 SNP 45% 2208 s52 SNP 75% 2173 s50 REF 60% 2209 s52 REF 80% 2174 s50 SNP 55% 2210 s52 SNP 55% 2175 s50 REF 60% 2211 s52 REF 60% 2176 s50 SNP 55% 2212 s52 REF 70% 2177 s50 SNP 55% 2213 s52 SNP 65% 2178 s50 REF 60% 2214 s52 REF 55% 2179 s50 REF 60% 2215 s52 SNP 50% 2180 s50 SNP 55% 2216 s52 SNP 70% 2181 s50 REF 60% 2217 s52 REF 75% 2182 s50 SNP 55% 2218 s52 SNP 65% 2183 s51 REF 65% 2219 s52 REF 50% 2184 s51 REF 50% 2220 s52 SNP 45% 2185 s52 REF 60% 2221 s52 SNP 65% 2186 s52 SNP 55% 2222 s52 REF 70% 2187 s52 REF 80% 2223 s53 REF 35% 2188 s52 SNP 75% 2224 s53 SNP 35% 2189 s52 SNP 45% 2225 s53 REF 35% 2190 s52 REF 75% 2226 s53 SNP 35% 2191 s52 SNP 70% 2227 s53 SNP 30% 2192 s52 REF 70% 2228 s53 REF 30% 2193 s52 REF 50% 2229 s53 REF 40% 2194 s52 SNP 45% 2230 s53 SNP 40% 2195 s52 REF 50% 2231 s53 SNP 35% 2196 s52 REF 60% 2232 s53 REF 35% 2197 s52 SNP 55% 2233 s53 SNP 55% 2198 s52 SNP 55% 2234 s53 REF 55% 2199 s52 REF 60% 2235 s53 SNP 45% 2200 s52 REF 60% 2236 s53 REF 45% 2201 s52 SNP 55% 2237 s53 SNP 35% 2202 s52 SNP 50% 2238 s53 REF 35% 2239 s53 REF 50% 2275 s54 SNP 45% 2240 s53 SNP 50% 2276 s54 SNP 60% 2241 s53 REF 45% 2277 s54 REF 55% 2242 s53 SNP 45% 2278 s54 REF 40% 2243 s53 SNP 30% 2279 s54 SNP 55% 2244 s53 REF 30% 2280 s54 REF 50% 2245 s53 REF 55% 2281 s54 REF 40% 2246 s53 SNP 40% 2282 s54 SNP 45% 2247 s53 REF 40% 2283 s55 REF 45% 2248 s53 SNP 50% 2284 s55 SNP 40% 2249 s53 REF 50% 2285 s55 SNP 40% 2250 s53 SNP 55% 2286 s55 REF 45% 2251 s53 SNP 35% 2287 s55 REF 50% 2252 s53 REF 35% 2288 s55 SNP 45% 2253 s54 REF 50% 2289 s55 REF 45% 2254 s54 SNP 55% 2290 s55 SNP 40% 2255 s54 REF 40% 2291 s55 REF 50% 2256 s54 SNP 45% 2292 s55 REF 45% 2257 s54 REF 55% 2293 s55 SNP 40% 2258 s54 SNP 45% 2294 s55 REF 55% 2259 s54 REF 40% 2295 s55 SNP 50% 2260 s54 SNP 60% 2296 s55 SNP 50% 2261 s54 REF 55% 2297 s55 REF 55% 2262 s54 SNP 60% 2298 s55 REF 50% 2263 s54 REF 55% 2299 s55 SNP 45% 2264 s54 SNP 60% 2300 s55 REF 55% 2265 s54 SNP 45% 2301 s55 SNP 50% 2266 s54 SNP 45% 2302 s55 REF 45% 2267 s54 REF 40% 2303 s55 SNP 40% 2268 s54 SNP 45% 2304 s55 SNP 45% 2269 s54 REF 40% 2305 s55 REF 50% 2270 s54 REF 50% 2306 s55 REF 50% 2271 s54 SNP 55% 2307 s55 SNP 45% 2272 s54 SNP 55% 2308 s55 SNP 45% 2273 s54 REF 50% 2309 s55 REF 50% 2274 s54 REF 40% 2310 s55 SNP 45% 2311 s55 SNP 40% 2347 s57 SNP 30% 2312 s55 REF 45% 2348 s57 REF 30% 2313 s55 SNP 45% 2349 s57 SNP 30% 2314 s55 REF 50% 2350 s57 REF 35% 2315 s56 REF 50% 2351 s57 REF 40% 2316 s56 SNP 45% 2352 s57 SNP 35% 2317 s56 SNP 30% 2353 s57 REF 30% 2318 s56 SNP 35% 2354 s57 SNP 45% 2319 s56 REF 40% 2355 s57 SNP 30% 2320 s56 REF 35% 2356 s57 REF 35% 2321 s56 REF 35% 2357 s57 REF 35% 2322 s56 SNP 30% 2358 s57 SNP 30% 2323 s56 SNP 50% 2359 s57 SNP 30% 2324 s56 REF 55% 2360 s57 REF 35% 2325 s56 SNP 30% 2361 s57 REF 50% 2326 s56 SNP 45% 2362 s57 REF 50% 2327 s56 REF 50% 2363 s57 SNP 45% 2328 s56 REF 55% 2364 s57 REF 30% 2329 s56 REF 35% 2365 s57 REF 35% 2330 s56 SNP 45% 2366 s57 SNP 30% 2331 s56 REF 50% 2367 s57 SNP 30% 2332 s56 SNP 35% 2368 s57 REF 35% 2333 s56 REF 40% 2369 s57 SNP 35% 2334 s56 REF 35% 2370 s57 REF 40% 2335 s56 SNP 30% 2371 s58 SNP 40% 2336 s56 SNP 50% 2372 s58 SNP 40% 2337 s56 REF 55% 2373 s58 REF 45% 2338 s56 REF 45% 2374 s58 SNP 45% 2339 s56 SNP 40% 2375 s58 REF 45% 2340 s56 SNP 50% 2376 s58 REF 45% 2341 s56 REF 40% 2377 s58 SNP 40% 2342 s56 SNP 35% 2378 s58 SNP 45% 2343 s57 REF 30% 2379 s58 SNP 45% 2344 s57 REF 30% 2380 s58 REF 50% 2345 s57 REF 30% 2381 s58 SNP 45% 2346 s57 REF 35% 2382 s58 REF 50% 2383 s58 REF 55% 2419 s60 SNP 65% 2384 s58 SNP 50% 2420 s60 REF 70% 2385 s58 REF 55% 2421 s60 SNP 60% 2386 s58 SNP 50% 2422 s60 REF 65% 2387 s58 SNP 45% 2423 s60 REF 65% 2388 s58 REF 50% 2424 s60 SNP 60% 2389 s58 SNP 50% 2425 s60 REF 65% 2390 s58 REF 55% 2426 s60 SNP 60% 2391 s58 REF 50% 2427 s60 REF 60% 2392 s58 SNP 45% 2428 s60 SNP 55% 2393 s58 REF 45% 2429 s60 REF 65% 2394 s58 SNP 40% 2430 s60 SNP 60% 2395 s58 SNP 50% 2431 s60 SNP 60% 2396 s58 REF 55% 2432 s60 REF 65% 2397 s58 REF 50% 2433 s60 REF 70% 2398 s58 SNP 45% 2434 s60 SNP 65% 2399 s58 REF 50% 2435 s60 SNP 60% 2400 s58 SNP 45% 2436 s60 REF 65% 2401 s58 SNP 45% 2437 s60 SNP 60% 2402 s58 SNP 45% 2438 s60 REF 65% 2403 s59 REF 50% 2439 s60 SNP 60% 2404 s59 SNP 55% 2440 s60 REF 65% 2405 s59 SNP 55% 2441 s60 REF 65% 2406 s59 SNP 55% 2442 s60 SNP 60% 2407 s59 REF 50% 2443 s61 SNP 60% 2408 s59 REF 50% 2444 s61 SNP 70% 2409 s59 SNP 55% 2445 s61 SNP 60% 2410 s59 SNP 55% 2446 s61 REF 70% 2411 s59 REF 50% 2447 s61 SNP 70% 2412 s59 SNP 55% 2448 s61 REF 60% 2413 s60 SNP 60% 2449 s61 REF 65% 2414 s60 REF 65% 2450 s61 SNP 65% 2415 s60 SNP 60% 2451 s61 REF 75% 2416 s60 REF 65% 2452 s61 SNP 75% 2417 s60 REF 65% 2453 s61 REF 60% 2418 s60 SNP 60% 2454 s61 REF 70% 2455 s61 SNP 60% 2491 s62 SNP 45% 2456 s61 SNP 60% 2492 s62 SNP 45% 2457 s61 SNP 60% 2493 s62 REF 50% 2458 s61 SNP 70% 2494 s62 SNP 50% 2459 s61 REF 65% 2495 s62 REF 55% 2460 s61 REF 70% 2496 s62 SNP 55% 2461 s61 REF 70% 2497 s62 SNP 45% 2462 s61 SNP 70% 2498 s62 SNP 50% 2463 s61 REF 60% 2499 s62 REF 50% 2464 s61 REF 65% 2500 s62 SNP 45% 2465 s61 REF 55% 2501 s63 REF 60% 2466 s61 SNP 55% 2502 s63 SNP 55% 2467 s61 SNP 65% 2503 s63 REF 55% 2468 s61 SNP 70% 2504 s63 SNP 50% 2469 s62 SNP 50% 2505 s63 SNP 50% 2470 s62 REF 50% 2506 s63 REF 55% 2471 s62 REF 45% 2507 s63 REF 50% 2472 s62 SNP 45% 2508 s63 SNP 45% 2473 s62 REF 50% 2509 s63 REF 55% 2474 s62 REF 50% 2510 s63 SNP 50% 2475 s62 SNP 45% 2511 s63 REF 60% 2476 s62 SNP 45% 2512 s63 SNP 55% 2477 s62 REF 55% 2513 s63 REF 55% 2478 s62 REF 45% 2514 s63 REF 60% 2479 s62 REF 55% 2515 s63 SNP 55% 2480 s62 REF 50% 2516 s63 REF 50% 2481 s62 SNP 50% 2517 s63 SNP 45% 2482 s62 SNP 50% 2518 s63 SNP 55% 2483 s62 REF 55% 2519 s63 SNP 55% 2484 s62 REF 50% 2520 s63 REF 60% 2485 s62 REF 55% 2521 s63 REF 60% 2486 s62 SNP 55% 2522 s63 SNP 55% 2487 s62 REF 55% 2523 s63 REF 60% 2488 s62 SNP 55% 2524 s63 SNP 50% 2489 s62 SNP 50% 2525 s63 REF 60% 2490 s62 REF 50% 2526 s63 SNP 55% 2527 s63 SNP 50% 2563 s65 SNP 50% 2528 s63 REF 55% 2564 s65 SNP 50% 2529 s64 REF 60% 2565 s65 REF 45% 2530 s64 SNP 55% 2566 s65 SNP 50% 2531 s64 SNP 45% 2567 s65 REF 45% 2532 s64 REF 50% 2568 s65 REF 45% 2533 s64 REF 55% 2569 s65 SNP 55% 2534 s64 SNP 50% 2570 s65 REF 50% 2535 s64 SNP 55% 2571 s65 REF 45% 2536 s64 REF 60% 2572 s65 SNP 50% 2537 s64 REF 50% 2573 s65 SNP 50% 2538 s64 SNP 45% 2574 s65 REF 45% 2539 s64 SNP 40% 2575 s86 SNP 35% 2540 s64 REF 45% 2576 s86 SNP 45% 2541 s65 REF 45% 2577 s86 REF 50% 2542 s65 SNP 50% 2578 s86 SNP 40% 2543 s65 SNP 50% 2579 s86 REF 45% 2544 s65 REF 45% 2580 s86 SNP 35% 2545 s65 REF 45% 2581 s86 REF 45% 2546 s65 SNP 50% 2582 s86 SNP 40% 2547 s65 SNP 50% 2583 s86 REF 45% 2548 s65 REF 45% 2584 s86 SNP 40% 2549 s65 REF 45% 2585 s86 REF 45% 2550 s65 SNP 50% 2586 s86 SNP 35% 2551 s65 REF 50% 2587 s86 REF 40% 2552 s65 SNP 55% 2588 s86 REF 45% 2553 s65 SNP 50% 2589 s86 REF 45% 2554 s65 REF 50% 2590 s86 REF 35% 2555 s65 SNP 55% 2591 s86 REF 45% 2556 s65 REF 55% 2592 s86 REF 30% 2557 s65 SNP 60% 2593 s86 SNP 40% 2558 s65 SNP 60% 2594 s86 REF 35% 2559 s65 REF 55% 2595 s86 SNP 40% 2560 s65 SNP 55% 2596 s86 REF 40% 2561 s65 REF 50% 2597 s86 SNP 35% 2562 s65 REF 45% 2598 s86 REF 40% 2599 s86 SNP 30% 2635 s67 SNP 45% 2600 s66 SNP 65% 2636 s67 SNP 35% 2601 s66 REF 70% 2637 s67 REF 30% 2602 s66 REF 75% 2638 s67 REF 45% 2603 s66 SNP 70% 2639 s67 SNP 50% 2604 s66 SNP 60% 2640 s67 SNP 40% 2605 s66 REF 65% 2641 s67 REF 35% 2606 s66 SNP 60% 2642 s67 SNP 35% 2607 s66 REF 65% 2643 s67 SNP 35% 2608 s66 REF 60% 2644 s67 REF 30% 2609 s66 SNP 55% 2645 s67 SNP 45% 2610 s66 SNP 60% 2646 s67 REF 40% 2611 s66 REF 65% 2647 s67 SNP 50% 2612 s66 REF 80% 2648 s67 REF 45% 2613 s66 SNP 75% 2649 s67 REF 40% 2614 s66 REF 70% 2650 s67 SNP 45% 2615 s66 SNP 65% 2651 s67 SNP 45% 2616 s66 SNP 55% 2652 s67 REF 40% 2617 s66 REF 60% 2653 s67 REF 30% 2618 s66 REF 60% 2654 s67 SNP 35% 2619 s66 SNP 55% 2655 s67 REF 30% 2620 s66 SNP 70% 2656 s67 SNP 35% 2621 s66 REF 75% 2657 s67 REF 30% 2622 s66 SNP 55% 2658 s68 SNP 50% 2623 s66 REF 60% 2659 s68 REF 55% 2624 s67 REF 35% 2660 s68 SNP 45% 2625 s67 SNP 40% 2661 s68 SNP 45% 2626 s67 SNP 50% 2662 s68 REF 50% 2627 s67 REF 45% 2663 s68 REF 60% 2628 s67 REF 40% 2664 s68 SNP 55% 2629 s67 SNP 35% 2665 s68 SNP 50% 2630 s67 REF 30% 2666 s68 REF 55% 2631 s67 SNP 45% 2667 s68 SNP 55% 2632 s67 REF 30% 2668 s68 REF 60% 2633 s67 SNP 35% 2669 s68 SNP 45% 2634 s67 REF 40% 2670 s68 SNP 45% 2671 s68 SNP 50% 2707 s69 REF 80% 2672 s68 REF 55% 2708 s69 SNP 75% 2673 s68 REF 55% 2709 s69 SNP 70% 2674 s68 SNP 50% 2710 s69 REF 75% 2675 s68 REF 50% 2711 s69 REF 70% 2676 s68 SNP 55% 2712 s69 SNP 65% 2677 s68 REF 60% 2713 s69 SNP 65% 2678 s68 REF 50% 2714 s70 SNP 45% 2679 s68 SNP 45% 2715 s70 REF 50% 2680 s68 REF 60% 2716 s70 SNP 45% 2681 s68 SNP 55% 2717 s70 REF 45% 2682 s68 REF 55% 2718 s70 SNP 40% 2683 s68 SNP 45% 2719 s70 REF 50% 2684 s68 REF 50% 2720 s70 REF 30% 2685 s68 REF 50% 2721 s70 REF 30% 2686 s68 SNP 45% 2722 s70 REF 50% 2687 s68 REF 50% 2723 s70 SNP 45% 2688 s68 SNP 45% 2724 s70 REF 30% 2689 s68 REF 50% 2725 s70 REF 40% 2690 s68 SNP 45% 2726 s70 SNP 35% 2691 s68 SNP 50% 2727 s70 SNP 30% 2692 s69 SNP 70% 2728 s70 REF 35% 2693 s69 REF 75% 2729 s70 REF 50% 2694 s69 SNP 70% 2730 s70 SNP 45% 2695 s69 SNP 70% 2731 s70 SNP 40% 2696 s69 REF 75% 2732 s70 REF 45% 2697 s69 REF 75% 2733 s70 SNP 35% 2698 s69 SNP 70% 2734 s70 REF 40% 2699 s69 REF 75% 2735 s70 REF 30% 2700 s69 SNP 70% 2736 s71 REF 75% 2701 s69 REF 75% 2737 s71 SNP 75% 2702 s69 REF 70% 2738 s71 SNP 75% 2703 s69 SNP 65% 2739 s71 SNP 75% 2704 s69 REF 70% 2740 s71 REF 75% 2705 s69 REF 75% 2741 s71 REF 75% 2706 s69 SNP 70% 2742 s71 REF 75% 2743 s71 SNP 75% 2779 s72 SNP 40% 2744 s71 REF 70% 2780 s72 REF 45% 2745 s71 SNP 70% 2781 s72 REF 45% 2746 s71 REF 75% 2782 s72 SNP 40% 2747 s71 SNP 75% 2783 s72 REF 45% 2748 s71 SNP 80% 2784 s72 SNP 40% 2749 s71 REF 80% 2785 s72 REF 50% 2750 s71 SNP 75% 2786 s72 SNP 40% 2751 s71 REF 75% 2787 s72 REF 45% 2752 s71 SNP 85% 2788 s72 REF 40% 2753 s71 REF 85% 2789 s72 REF 50% 2754 s71 REF 75% 2790 s72 SNP 45% 2755 s71 SNP 75% 2791 s72 SNP 45% 2756 s71 SNP 70% 2792 s72 REF 50% 2757 s71 REF 70% 2793 s72 REF 45% 2758 s71 REF 75% 2794 s72 REF 45% 2759 s71 SNP 75% 2795 s72 REF 45% 2760 s71 REF 70% 2796 s72 REF 45% 2761 s71 SNP 70% 2797 s72 SNP 40% 2762 s72 SNP 35% 2798 s72 REF 45% 2763 s72 REF 40% 2799 s72 SNP 40% 2764 s72 SNP 35% 2800 s73 SNP 55% 2765 s72 REF 45% 2801 s73 SNP 50% 2766 s72 SNP 40% 2802 s73 REF 55% 2767 s72 SNP 40% 2803 s73 REF 60% 2768 s72 REF 45% 2804 s73 REF 50% 2769 s72 SNP 45% 2805 s73 SNP 45% 2770 s72 REF 50% 2806 s73 SNP 50% 2771 s72 SNP 40% 2807 s73 REF 55% 2772 s72 REF 45% 2808 s73 REF 60% 2773 s72 SNP 45% 2809 s73 SNP 55% 2774 s72 REF 50% 2810 s73 REF 55% 2775 s72 SNP 40% 2811 s73 SNP 50% 2776 s72 REF 45% 2812 s73 REF 55% 2777 s72 REF 50% 2813 s73 REF 55% 2778 s72 REF 45% 2814 s73 SNP 50% 2815 s73 SNP 55% 2851 s74 REF 40% 2816 s73 REF 60% 2852 s74 REF 40% 2817 s73 SNP 55% 2853 s74 SNP 35% 2818 s73 REF 60% 2854 s74 REF 45% 2819 s73 SNP 50% 2855 s74 SNP 40% 2820 s73 REF 55% 2856 s75 SNP 45% 2821 s73 REF 55% 2857 s75 REF 40% 2822 s73 SNP 50% 2858 s75 REF 35% 2823 s73 REF 60% 2859 s75 SNP 40% 2824 s73 SNP 55% 2860 s75 REF 45% 2825 s73 REF 55% 2861 s75 SNP 50% 2826 s73 SNP 50% 2862 s75 SNP 30% 2827 s73 SNP 45% 2863 s75 SNP 50% 2828 s73 REF 50% 2864 s75 REF 45% 2829 s73 SNP 50% 2865 s75 REF 45% 2830 s74 SNP 40% 2866 s75 SNP 50% 2831 s74 REF 45% 2867 s75 SNP 55% 2832 s74 SNP 35% 2868 s75 REF 50% 2833 s74 REF 40% 2869 s75 REF 40% 2834 s74 REF 50% 2870 s75 SNP 45% 2835 s74 SNP 45% 2871 s75 SNP 35% 2836 s74 REF 55% 2872 s75 REF 30% 2837 s74 SNP 50% 2873 s75 SNP 45% 2838 s74 SNP 40% 2874 s75 REF 40% 2839 s74 REF 45% 2875 s75 SNP 50% 2840 s74 SNP 45% 2876 s75 REF 45% 2841 s74 REF 50% 2877 s75 REF 30% 2842 s74 REF 45% 2878 s75 SNP 35% 2843 s74 SNP 45% 2879 s75 SNP 30% 2844 s74 REF 50% 2880 s75 REF 50% 2845 s74 SNP 40% 2881 s75 SNP 55% 2846 s74 SNP 40% 2882 s75 SNP 55% 2847 s74 REF 45% 2883 s75 REF 50% 2848 s74 REF 40% 2884 s75 REF 50% 2849 s74 SNP 35% 2885 s75 SNP 55% 2850 s74 SNP 35% 2886 s75 REF 40% 2887 s75 SNP 45% 2923 s76 REF 55% 2888 s75 SNP 30% 2924 s76 SNP 50% 2889 s87 SNP 60% 2925 s76 REF 45% 2890 s87 SNP 65% 2926 s76 REF 50% 2891 s87 SNP 65% 2927 s76 SNP 45% 2892 s87 SNP 60% 2928 s76 REF 45% 2893 s87 SNP 65% 2929 s76 SNP 40% 2894 s87 SNP 65% 2930 s76 SNP 50% 2895 s87 SNP 70% 2931 s76 REF 55% 2896 s87 SNP 60% 2932 s76 SNP 40% 2897 s87 REF 55% 2933 s76 REF 45% 2898 s87 SNP 65% 2934 s76 SNP 40% 2899 s87 REF 45% 2935 s77 SNP 60% 2900 s87 SNP 50% 2936 s77 REF 65% 2901 s87 SNP 65% 2937 s77 REF 80% 2902 s87 SNP 65% 2938 s77 SNP 75% 2903 s87 SNP 65% 2939 s77 SNP 75% 2904 s87 SNP 50% 2940 s77 SNP 45% 2905 s76 SNP 45% 2941 s77 REF 50% 2906 s76 SNP 45% 2942 s77 REF 55% 2907 s76 REF 50% 2943 s77 SNP 50% 2908 s76 SNP 50% 2944 s77 REF 70% 2909 s76 REF 55% 2945 s77 SNP 65% 2910 s76 SNP 45% 2946 s77 SNP 75% 2911 s76 REF 50% 2947 s77 REF 50% 2912 s76 SNP 45% 2948 s77 SNP 45% 2913 s76 REF 50% 2949 s77 REF 40% 2914 s76 SNP 45% 2950 s77 REF 80% 2915 s76 REF 50% 2951 s77 SNP 75% 2916 s76 REF 50% 2952 s77 REF 80% 2917 s76 SNP 45% 2953 s77 SNP 35% 2918 s76 REF 50% 2954 s77 REF 65% 2919 s76 SNP 45% 2955 s77 SNP 60% 2920 s76 REF 50% 2956 s77 SNP 60% 2921 s76 REF 55% 2957 s77 REF 65% 2922 s76 SNP 50% 2958 s77 REF 80% 2959 s77 SNP 75% 2985 s78 SNP 40% 2960 s77 REF 80% 2986 s78 REF 45% 2961 s77 SNP 75% 2987 s78 REF 35% 2962 s77 SNP 50% 2988 s78 SNP 30% 2963 s77 REF 55% 2989 s78 SNP 40% 2964 s77 REF 55% 2990 s78 REF 45% 2965 s77 SNP 50% 2991 s78 REF 45% 2966 s77 REF 65% 2992 s78 SNP 40% 2967 s77 SNP 60% 2993 s78 REF 45% 2968 s77 REF 80% 2994 s78 SNP 45% 2969 s77 SNP 35% 2995 s78 REF 50% 2970 s77 REF 40% 2996 s78 SNP 40% 2971 s77 SNP 65% 2997 s78 REF 45% 2972 s77 REF 70% 2998 s78 REF 50% 2973 s77 SNP 50% 2999 s78 SNP 35% 2974 s77 REF 55% 3000 s78 SNP 45% 2975 s78 REF 40% 3001 s78 REF 40% 2976 s78 SNP 30% 3002 s78 SNP 40% 2977 s78 SNP 35% 3003 s78 REF 45% 2978 s78 REF 35% 3004 s78 REF 40% 2979 s78 SNP 40% 3005 s78 SNP 35% 2980 s78 REF 45% 3006 s78 SNP 30% 2981 s78 REF 45% 3007 s78 SNP 40% 2982 s78 SNP 40% 3008 s78 REF 35% 2983 s78 SNP 35% 3009 s78 REF 45% 2984 s78 REF 40% 3010 s78 SNP 40%

Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.

EXPERIMENTAL DETAILS Example 1: BEST1 Correction Analysis

Guide sequences comprising 17-20 nucleotides in the sequences of 17-20 contiguous nucleotides set forth in SEQ ID NOs: 1-3010 are screened for high on target activity. On target activity is determined by DNA capillary electrophoresis analysis.

According to DNA capillary electrophoresis analysis, guide sequences comprising 17-20 nucleotides in the sequences of 17-20 contiguous nucleotides set forth in SEQ ID NOs: 1-3010 are found to be suitable for correction of the BEST1 gene.

DISCUSSION

The guide sequences of the present invention are determined to be suitable for targeting the BEST1 gene.

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What is claimed is:
 1. A method for inactivating a mutant Bestrophin 1 (BEST1) allele in a cell comprising a mutant BEST1 allele and a functional BEST1 allele, the method comprising delivering to the cell a composition comprising a) a first RNA molecule which comprises a guide sequence portion having 17-24 nucleotides and which targets a rs1800009 single nucleotide polymorphism (SNP) position in the mutant BEST1 allele; b) a second RNA molecule which comprises a guide sequence portion having 17-24 nucleotides and which targets a sequence present in an intron of both the mutant BEST1 allele and the functional BEST1 allele; and c) an RNA guided CRISPR nuclease, wherein the first and second RNA molecules either (1) are guide RNA molecules comprising a portion having a sequence which binds to a CRISPR nuclease, or (2) comprise a portion having a tracr mate sequence and the composition further comprises a tracrRNA molecule that hybridizes with the tracr mate sequence, wherein the sequence of the rs1800009 SNP position in the mutant BEST1 allele differs from the sequence of the rs1800009 SNP position in the functional BEST1 allele, wherein the first RNA molecule and the RNA guided CRIPSR nuclease form a complex that creates a double strand break in the mutant BEST1 allele, wherein the second RNA molecule and the RNA guided CRIPSR nuclease form a complex that creates a double strand break in an intron of both the mutant BEST1 allele and the functional BEST1 allele, and wherein a portion of the mutant BEST1 allele is excised.
 2. The method of claim 1, wherein the first and/or second RNA molecules are guide RNA molecules comprising a portion having a sequence which binds to a CRISPR nuclease, wherein said sequence is a tracrRNA sequence.
 3. The method of claim 1, wherein the first and/or second RNA molecules comprise one or more linker portions.
 4. The method of claim 1, wherein the first and/or second RNA molecules are up to 300 nucleotides in length.
 5. The method of claim 1, wherein the first and/or second RNA molecules comprise a portion having a tracr mate sequence and the composition comprises a tracrRNA molecule.
 6. The method of claim 1, wherein the sequence of the 17-24 nucleotides of the guide sequence portion of the second RNA molecule is different than sequence of the 17-24 nucleotides of the guide sequence portion of the first RNA molecule.
 7. The method of claim 1, wherein the inactivating results in a truncated protein encoded by the mutated allele and a functional protein encoded by the functional allele.
 8. The method of claim 1, wherein the RNA guided CRISPR nuclease utilizes a NGG protospacer adjacent motif (PAM).
 9. The method of claim 1, wherein the RNA guided CRISPR nuclease is a Streptococcus pyogenes Cas9 nuclease or a Staphylococcus aureus Cas9 nuclease.
 10. The method of claim 8, wherein the guide sequence portion of the first RNA molecule comprises 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 102-109 and 960-991.
 11. The method of claim 8, wherein the guide sequence portion of the first RNA molecule comprises 17-20 contiguous nucleotides set forth in SEQ ID NO: 108 or SEQ ID NO:
 109. 